Compounds and methods for modulating frataxin expression

ABSTRACT

The present technology relates to compositions and methods for modulating expression of genes, which include a target oligonucleotide sequence, such as repeats of a particular oligonucleotide sequence containing 3 to 10 nucleotides. In particular aspects, the present technology relates to agents having a formula A-L-B, wherein -L- is a linker; A- is a Brd4 binding moiety; and -B is a nucleic acid binding moiety, such as a polyamide or complementary oligonucleotide, that specifically binds to the target oligonucleotide sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. provisional patentapplication U.S. Ser. No. 62/315,466, filed Mar. 30, 2016, and U.S.provisional patent application U.S. Ser. No. 62/366,700, filed Jul. 26,2016, which are incorporated by reference herein in their entireties.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under CA133508 andHL099773 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 24, 2017, isnamed 999400-5743_SL.TXT and is 11,740 bytes in size.

BACKGROUND

Friedreich's ataxia (also referred to as FA or FRDA) is a rare but fatalautosomal recessive neurodegenerative disease, with an estimatedincidence of 1 in every 40,000 people. This condition is typically foundin individuals with European, Middle Eastern, or North African ancestry.FRDA causes progressive damage to the nervous system and muscle cells,resulting in a loss of coordination as well as various neurological andcardiac complications. In particular, FRDA patients developneurodegeneration of the large sensory neurons and spinocerebellartracts, as well as cardiomyopathy and diabetes mellitus. Onset ofsymptoms is typically seen between the ages of 5 and 15 years, and themean age of death is approximately 38 years.

Friedreich's ataxia is caused by an abnormal expansion of theguanine-adenine-adenine (GAA) trinucleotide repeat sequences in intron 1of the frataxin (FXN) gene, resulting in transcriptional repression andreduced expression of the frataxin (FXN) protein. Frataxin, which isencoded by the nuclear frataxin (FXN) gene, is a highly-conserved,210-amino acid protein that is localized to the mitochondrion. Most FRDApatients (approximately 98%) carry a homozygous mutation characterizedby an expansion of a GAA trinucleotide repeat in the first intron of thefrataxin (FXN) gene. Pathological GAA expansions can range from about 66to more than 1,000 trinucleotide repeats, whereas frataxin alleles thatare not associated with FRDA comprise from about 6 to about 34 repeats.

There is presently no cure for FRDA or specific therapy to preventprogression of the disease which has been approved for use as atreatment. Therefore, there is a need to develop compositions thatrestore or partially restore frataxin levels to treat and/or preventFRDA.

SUMMARY

The present technology relates generally to compositions and methods formodulating expression of genes which include a target oligonucleotidesequence, e.g., typically a specific oligonucleotide sequence containingabout 10 to 100 nucleotides. In particular aspects, the presenttechnology relates to agents having a formula A-L-B, wherein -L- is alinker, typically a covalent linker having a backbone chain including atleast 10 atoms; A- is a Brd4 binding moiety; and -B is a nucleic acidbinding moiety that specifically binds to a target oligonucleotidesequence, e.g., a polyamide that specifically binds to one or morerepeats of a GAA oligonucleotide sequence, or an oligonucleotidesequence (e.g., containing about 15 to 30 nucleotides) that iscomplementary to a desired target oligonucleotide sequence.

In some aspects, the present technology relates to compositions andmethods for modulating expression of genes which include repeats of anoligonucleotide sequence containing 3 to 6 nucleotides, such as a GAAoligonucleotide sequence. In particular aspects, the nucleic acidbinding moiety (-B) is a polyamide that specifically binds to one ormore repeats of a GAA oligonucleotide sequence.

Disclosed herein are methods and compositions for modulating geneexpression. In one aspect, the compositions comprise any one or more ofthe agents shown in Section II. In some embodiments, the agent has aformula A-L-B, wherein -L- is a linker; A- is a Brd4 binding moiety; and-B is polyamide that specifically binds to one or more repeats of anoligonucleotide sequence containing 3 to 6 nucleotides, such as a GAAoligonucleotide sequence.

In some embodiments, A may be a triazolodiazepine Brd4 binding moiety orrelated structure, such as a thienotriazolodiazepine Brd4 bindingmoiety.

In some embodiments, the agent is capable of increasing mRNA expressionlevels of a gene which includes repeats of a GAA oligonucleotidesequence, e.g., increasing frataxin mRNA levels in a cell derived from aFriedreich's ataxia (FRDA) patient. In some embodiments, the agent iscapable of increasing frataxin mRNA levels in a GM15850 FRDA patientcell line relative to an untreated GM15850 cell. In some embodiments,the agent is capable of inducing at least about a 2-fold increase infrataxin mRNA levels in a GM15850 FRDA patient cell line relative to anuntreated GM15850 cell line. In some embodiments, the agent is capableof inducing at least about a 2.5-fold, 3-fold, or 3.5-fold increase infrataxin mRNA levels in a GM15850 FRDA patient cell line relative to anuntreated GM15850 cell line. In some embodiments, the agent is capableof inducing at least about a 4-fold increase in frataxin mRNA levels ina GM15850 FRDA patient cell line relative to an untreated GM15850 cellline. In some embodiments, the agent is capable of inducing at leastabout a 6-fold increase in frataxin mRNA levels in a GM15850 FRDApatient cell line relative to an untreated GM15850 cell line. In someembodiments, the agent is capable of inducing at least about an 8-foldincrease in frataxin mRNA levels in a GM15850 FRDA patient cell linerelative to an untreated GM15850 cell line. In some embodiments, theagent is capable of inducing at least about a 2.5-fold increase, atleast about a 4-fold increase, at least about a 6-fold increase, or atleast about an 8-fold increase in frataxin mRNA levels in a GM15850 FRDApatient cell line relative to an untreated GM15850 cell line.

In one aspect, the present disclosure provides a pharmaceuticalcomposition comprising a therapeutically effective amount of any one ormore of the agents described in Section II and a pharmaceuticallyacceptable carrier. In some embodiments the pharmaceutically acceptablecarrier is selected from one or more of saline, solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents compatible with pharmaceuticaladministration. In some embodiments, the therapeutically effectiveamount of the agent is between 0.1 mg/kg to about 7.5 mg/kg body weightof a subject in need thereof.

In one aspect, the present disclosure provides a method for modulatingtranscription of a gene that includes multiple repeats of anoligonucleotide sequence containing 3 to 6 nucleotides, such as a GAAoligonucleotide repeat expansion. Without wishing to be bound by theory,the modulation of transcription is effected by contacting the gene withan agent of the present technology having a formula A-L-B, wherein -L-is a linker; A- is a Brd4 binding moiety; and -B is a polyamide thatspecifically binds to one or more repeats of the oligonucleotidesequence, thereby modulating the transcription of the gene.

In one aspect, the present disclosure provides a method for increasingfrataxin mRNA levels in a cell comprising contacting the cell with aneffective amount of any one or more of the agents shown in Section II.In another aspect, the present disclosure provides a method forincreasing frataxin protein levels in a cell, comprising contacting thecell with an effective amount of any one or more of the agents shown inSection II. In some embodiments, the cell may be derived from aFriedreich's ataxia patient. In some embodiments, the cell may bederived from a Friedreich's ataxia patient cell line. In someembodiments, the Friedreich's ataxia patient cell line is a GM15850 cellline. In some embodiments, the cell may be a dorsal root ganglia neuron,cardiomyocyte, pancreatic beta cell, peripheral blood mononuclear cell(PBMC), B-lymphocyte, lymphoblastoid cell, and/or fibroblast.

In some embodiments, the cell comprises a gene associated with a geneticcondition comprising at least about 30 repeats, and in some instances atleast about 50 repeats of an oligonucleotide sequence having 3 to 6nucleotides. In some embodiments, the cell comprises a gene associatedwith a genetic condition comprising at least about 70 repeats of theoligonucleotide sequence. In some embodiments, the cell comprises a geneassociated with a genetic condition comprising at least about 100repeats of the oligonucleotide sequence. In some embodiments, the cellcomprises a gene associated with a genetic condition comprising at leastabout 200 repeats of the oligonucleotide sequence.

In some embodiments, the cell comprises a frataxin (FXAN) gene includingat least about 50 GAA repeats. In some embodiments, the cell comprisesan FXN gene including at least about 70 GAA repeats. In someembodiments, the cell comprises an FXN gene including at least about 100GAA repeats. In some embodiments, the cell comprises an FXN geneincluding at least about 200 GAA repeats.

In some embodiments, the frataxin mRNA levels are increased within about6 hours hours after contacting the cell with any one or more of theagents shown in Section II. In some embodiments, the frataxin mRNAlevels are increased within about 24 hours after contacting the cellwith any one or more of the agents shown in Section II. In someembodiments, the frataxin mRNA levels are increased within about 2 daysafter contacting the cell with any one or more of the agents shown inSection II. In some embodiments, the frataxin mRNA levels are increasedwithin about 3 days after contacting the cell with any one or more ofthe agents shown in Section II.

In one aspect, the present disclosure provides a method for treatingFriedreich's ataxia (FRDA) in a subject in need thereof, comprisingadministering any one or more of the agents shown in Section II. In someembodiments, the present disclosure provides a method for increasingfrataxin mRNA levels in the subject. In some embodiments, frataxin mRNAlevels of the subject are increased relative to those in the subjectprior to treatment. In some embodiments, the frataxin mRNA levels areincreased by at least about 2.5-fold. In some embodiments, the frataxinmRNA levels are increased by at least about 4-fold. In some embodiments,the frataxin mRNA levels are increased by at least about 8-fold. In someembodiments, frataxin protein levels of the subject are increasedrelative to those in the subject prior to treatment.

In some embodiments, the treatment comprises ameliorating one or moresymptoms of Friedreich's ataxia. In some embodiments the symptoms ofFriedreich's ataxia comprise one or more of ataxia, gait ataxia, muscleweakness, loss of coordination, loss of balance, lack of reflexes inlower limbs, loss of tendon reflexes, loss of ability to feel vibrationsin lower limbs, loss of sensation in the extremities, loss of upper bodystrength, weakness in the arms, spasticity, loss of tactile sensation,impairment of position sense, impaired perception of light touch,impaired perception of pain, impaired perception of temperature, visionimpairment, color vision changes, involuntary eye movements, pes cavus,inversion of the feet, hearing impairment, dysarthria, dysphagia,impaired breathing, scoliosis, diabetes, glucose intolerance,carbohydrate intolerance, hypertrophic cardiomyopathy, arrhythmia,myocardial fibrosis, cardiac failure, elevated serum or plasma highsensitive troponin-T (hsTNT) (>14 ng/L), or reduced serum or plasmafrataxin protein levels (≤19 ng/mL for pediatric and ≤21 ng/mL for adultpatients).

In some embodiments, the subject is human.

In some embodiments, the agent is administered orally, topically,systemically, intravenously, subcutaneously, transdermally,iontophoretically, intranasally, intraperitoneally, or intramuscularly.

In some embodiments, the present disclosure provides a method fortreating Friedreich's ataxia (FRDA) in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising any one or more of theagents of Section II and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are bar graphs illustrating the use of the agent of thepresent technology to increase frataxin levels in FRDA patient cells.FIG. 1A shows relative FXN mRNA levels produced in the GM15850Friedreich's ataxia patient derived cell line following incubation for24 hours with a control solution (0.1% DMSO), Control polyamide 3 (“3”),JQ1-(S), a mixture of Control polyamide 3 and JQ1-(S), Control conjugate2 (“2”), and varying concentrations of Agent 4 (“4”). FIG. 1B showsrelative FXN mRNA levels produced in a cell line that was derived from aclinically unaffected individual having two FXN alleles in the normalrange of GAA trinucleotide repeats (GM15851) following incubation for 24hours with a control solution (0.1% DMSO), Control polyamide 3 (“3”),JQ1-(S), a mixture of Control polyamide 3 and JQ1-(S), Control conjugate2 (“2”), and varying concentrations of Agent 4 (“4”). FIG. 1C shows aside-by-side comparison of the data illustrated by FIGS. 1B and 1A.

FIG. 2 is a bar graph showing luciferase activity of treated reportercell lines FXN-Luc and FXN-GAA-Luc treated with Agent 4 and variouscontrols, including Polyamide 3 (“3”), JQ1, and Compound 109.

FIGS. 3A and 3B show immunoblots for FXN and α-tubulin (TUB) in GM15850cells (FIG. 3A) or GM15851 cells (FIG. 3B) treated with varyingconcentrations of Control Conjugate 2 (“2”), Polyamide 3 (“3”), JQ1, orAgent 4 (“4”).

FIG. 4 is a chart showing relative RNA concentrations of GM15850 cellsharvested after 24 hours of treatment with the described compounds.

FIG. 5 is an image of a PyMOL structure modeling the design of thelinker of the present technology (PEG6). The PyMOL structure shows apolyamide bound to a nucleosome and JQ1-(S) bound to Brd4. The minimaldistance between these structures is measured and the length of thelinker is shown. Two different .pdb files were opened in the same windowand brought together to illustrate the importance of -L in the design ofthe full molecule.

FIG. 6 is a bar graph showing relative frataxin (FXN) mRNA levelsproduced in lymphoblastoid cell lines derived from three FRDA patients(P1-P3) following incubation for 24 hours with 1 μM Control polyamide 3and unbound JQ1 (“3+JQ1”) or 1 μM Agent 4 (“4”).

FIG. 7 is a bar graph showing relative frataxin (FXN) mRNA levelsproduced in primary samples of peripheral blood mononuclear cells(PBMCs) derived from eleven FRDA patients (P1-P11) following incubationfor 24 hours with 1 μM Agent 4 (“4”).

FIG. 8 is a chart showing the results of an AlphaScreen™ assay of thebinding of Control polyamide 3 (“3”), Agent 4 (“4”), Control polyamide 1(“1”), Control polyamide 1 bound to JQ1 without a linker (“1-JQ1”), andunconjugated JQ1 (“JQ1-(S)”) to Bdr4.

FIGS. 9A-9C show ChIP-seq data plots of read density over the entirefrataxin (FXN) gene body (FIG. 9D) for Brd4 (FIGS. 9A-9C) followingincubation for 24 hours with a control solution (0.1% DMSO, FIG. 9A) or1 μM Control polyamide 3 and unbound JQ1 (“3+JQ1”, FIG. 9B) or 1 μMAgent 4 (“4”, FIG. 9C).

FIGS. 10A-10C show ChIP-seq data plots of read density over the entirefrataxin (FXN) gene body (FIG. 10D) for pSer2 (phosphorylated Ser2 ofthe carboxy-terminal domain (CTD) of RNA polymerase II) (FIGS. 10A-10C)following incubation for 24 hours with a control solution (0.1% DMSO,FIG. 10A) or 1 μM Control polyamide 3 and unbound JQ1 (“3+JQ1”, FIG.10B) or 1 μM Agent 4 (“4”, FIG. 10C).

FIGS. 11A-11C show ChIP-seq data plots of read density over the entirefrataxin (FXN) gene body (FIG. 11D) for pol2 (RNA polymerase II) (FIGS.11A-11C) following incubation for 24 hours with a control solution (0.1%DMSO, FIG. 11A) or 1 μM Control polyamide 3 and unbound JQ1 (“3+JQ1”,FIG. 11B) or 1 μM Agent 4 (“4”, FIG. 11C).

FIG. 12 is a schematic depicting a model transcription elongationstimulator.

FIG. 13 depicts a crystal structure showing (S)-JQ1 bound to Brd4(Protein Data Bank accession 3MXF). The tert-butyl group, highlighted byan asterisk, projects out of the binding pocket of Brd4, indicating thatchemical substitution at this position would likely be tolerated.

FIG. 14 is a schematic depicting examples of compounds (includingcontrols) tested by the methods of the present technology to determinetheir ability to function as transcription elongation stimulatorsincluding Polyamide 1 (“1”) Control Conjugate 2 (“2”), Polyamide 3 (“3”)and Agent 4 (“4”).

DETAILED DESCRIPTION

The present disclosure relates to compositions and methods formodulating the expression of genes which include repeats of anoligonucleotide sequence containing 3 to 6 nucleotides, such as a GAAoligonucleotide sequence. Many embodiments of the methods are directedto modulating frataxin (FXN) gene expression, and for treatingFriedreich's ataxia. Disclosed herein are agents having a formula A-L-B,wherein -L- is a linker; A- is a Brd4 binding moiety; and -B is anucleic acid binding moiety, such as a polyamide that specifically bindsto one or more repeats of a GAA oligonucleotide sequence. Also disclosedherein are methods for increasing frataxin (FXN) mRNA levels in a cellcomprising contacting the cell with an effective amount of one or moreof the agents. Also disclosed herein are methods for increasing frataxin(FXN) protein levels in a cell, comprising contacting the cell with aneffective amount of one or more of the agents. Also disclosed herein aremethods of treating Friedreich's ataxia (FRDA) in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of one or more of the agents.

I. Friedreich's Ataxia

Friedreich's ataxia (FA or FRDA) is an autosomal recessiveneurodegenerative disorder caused by mutations in the FXN gene, whichencodes the protein frataxin. Human frataxin is synthesized as a210-amino acid precursor that is localized to the mitochondrion wherethe protein is subsequently cleaved to a mature 14 kDa protein (aminoacid residues 81-210). FRDA is caused by a hyper-expansion of GAArepeats in the first intron of the FXN gene, resulting intranscriptional repression and insufficient expression of frataxin(FXN), a highly-conserved, iron-binding mitochondrial protein.Transcription is a multistep, highly-regulated process that is dividedinto three stages: initiation, elongation, and termination. Withoutwishing to be bound by any particular theory, recent evidence suggeststhat transcriptional elongation is the primary step affected by thepathological GAA expansion, with the expanded GAA repeats leading to thepremature termination or pausing of FXNtranscription and, ultimately,decreased cellular frataxin protein levels. Accordingly, without wishingto be bound by any particular theory, FRDA may be characterized as atranscriptional pausing-based genetic disease caused by a defect intranscriptional elongation resulting in transcriptional repression andreduced expression of a gene (e.g., FXN). RNA polymerase-II initiatestranscription of the repressed gene underlying the disease, but fails toelongate through the entire open reading frame of the gene to producefull-length pre-mRNA. Splicing is typically unaffected, thereby allowingfor the production of normal full-length protein, albeit at reducedlevels.

Friedreich's ataxia is the most common hereditary ataxia and causesprogressive damage to the nervous system, particularly sensory neurons.Although frataxin is ubiquitously expressed, certain cells (e.g., dorsalroot ganglia neurons, cardiomyocytes, and pancreatic beta cells) areparticularly sensitive to frataxin depletion, and the resultingdegenerative loss of these cells accounts for the clinicalmanifestations of FRDA. FRDA patients develop neurodegeneration of thelarge sensory neurons and spinocerebellar tracts, as well ascardiomyopathy and diabetes mellitus. Clinical symptoms of FRDA includeataxia, gait ataxia, muscle weakness, loss of coordination, loss ofbalance, lack of reflexes in lower limbs, loss of tendon reflexes, lossof ability to feel vibrations in lower limbs, loss of sensation in theextremities, loss of upper body strength, weakness in the arms,spasticity, loss of tactile sensation, impairment of position sense,impaired perception of light touch, impaired perception of pain,impaired perception of temperature, vision impairment, color visionchanges, involuntary eye movements, pes cavus, inversion of the feet,hearing impairment, dysarthria, dysphagia, impaired breathing,scoliosis, diabetes, glucose intolerance, carbohydrate intolerance,hypertrophic cardiomyopathy, arrhythmia, myocardial fibrosis, cardiacfailure, elevated serum or plasma high sensitive troponin-T (hsTNT) (>14ng/L), and reduced serum or plasma frataxin protein levels (≤19 ng/mLfor pediatric and ≤21 ng/mL for adult patients).

There is an inverse correlation between the number of GAA repeats andFXN protein levels, and there is a tight correlation between frataxinprotein levels and the severity of disease. That is, lower frataxinprotein levels correlate with greater numbers of GAA repeats and diseaseseverity. FRDA patients exhibiting clinical symptoms have frataxinprotein levels that are between 5% and 35% those of healthy individuals.Asymptomatic heterozygous carriers have frataxin mRNA and protein levelsthat are about 40-50% those of healthy individuals. Most FRDA patients(approximately 98%) carry a homozygous mutation in the first intron ofthe frataxin (FXN) gene comprising an expansion of a GAA trinucleotiderepeat. Pathological GAA expansions can range from about 66 to more than1,000 trinucleotide repeats, whereas frataxin alleles that are notassociated with disease comprise from about 6 to about 34 repeats. Veryrare cases of FRDA (about 4%) are characterized by an expansion of a GAAtrinucleotide repeat present in one allele and a deleterious pointmutation in the other allele. It is generally understood that longer GAAtrinucleotide repeats are associated with greater deficiency of frataxinand earlier onset and increased severity of disease. Partially restoringfrataxin in affected cells may slow or prevent disease progression.

Diagnosis

FRDA is diagnosed by assessing clinical criteria and/or performinggenetic testing (Wood, N. W., Arch. Dis. Child., 78:204-207 (1998)). Thepatient's medical history is evaluated and a physical examinationperformed. Key to diagnosing FRDA is the recognition of hallmarksymptoms, including balance difficulty, loss of joint sensation, absenceof reflexes, and signs of neurological problems. In addition, genetictesting can provide a conclusive diagnosis of FRDA.

Clinical Criteria. Strict clinical criteria have been developed that arewidely used in the diagnosis of FRDA (Harding, A. E., Brain, 104:589-620(1981)). Diagnostic criteria include an age of onset before 25 years ofage, as well as presence of the following symptoms: progressive ataxiaof gait and limbs, absence of knee and ankle jerks, axonal picture onneurophysiology, and dysarthria (if after five years from onset). Inover 66% of individuals with FRDA, the following symptoms are present:scoliosis, pyramidal weakness in lower limbs, absence of reflexes inarms, large fibre sensory loss on examination, and abnormal ECG. In lessthan 50% of individuals having FRDA, the following symptoms are present:nystagmus, optic atrophy, deafness, distal amyotrophy, pes cavus, anddiabetes. However, some cases of FRDA present atypically. For example,onset of FRDA may occur over the age of 20 years in some patients.Moreover, some patients retain tendon reflexes.

Core features of pyramidal tract involvement include the association ofextensor plantar responses, absence of ankle reflexes, and a progressivecourse of disease. Pyramidal weakness in lower limbs can lead toparalysis. Skeletal abnormalities are common in FRDA. For example,scoliosis is present in approximately 85% of FRDA patients. Footabnormalities may be present, including pes cavus and, less frequently,pes planus and equinovarus. Amyotrophy of the lower legs may occur.Optic atrophy is present in about 25% of FRDA cases, while major visualimpairment occurs in less than 5% of cases. Deafness is present in lessthan 10% of FRDA cases. Blood sugar analysis is also performed, asdiabetes is seen in approximately 10% of FRDA patients. About 20% ofFRDA patients develop carbohydrate intolerance.

A prominent non-neurological feature of FRDA is cardiomyopathy, whichmay initially present as the sole symptom of disease. Anelectrocardiogram (ECG) may be performed to assess electrical andmuscular functions of the heart. Approximately 65% of FRDA patientspresent with an abnormal ECG, having widespread T wave inversion in theinferolateral chest leads. The most frequent echocardiographicabnormality in FRDA patients is concentric ventricular hypertrophy.Heart failure typically occurs late in disease progression, oftenaccounting for premature death in FRDA patients.

Within a few years after onset of FRDA, the patient presents withdysarthria and pyramidal weakness, and subsequent nystagmus, which ischaracterized by involuntary repetitive and jerky eye movements. Withinabout 10-15 years after onset of disease, the patient becomes wheelchairbound.

Additional tests typically employed to assess FRDA patients includeelectromyogram (EMG) to measure electrical activity of muscle cells,nerve conduction studies to measure nerve impulse transmission speed,echocardiogram to record the position and motion of heart muscle, andblood tests to determine if the patient has vitamin E deficiency.Magnetic resonance imaging (MRI) or computed tomography (CT) scansprovide brain and spinal cord images that can be useful to rule outother neurological conditions.

Genetic Testing. FRDA is a neurological disorder caused by mutations inthe frataxin (FXN) gene, having a cytogenetic location of 9q21.11.DNA-based testing is one method that is used to diagnose FRDA.Homozygosity for a GAA repeat expansion in intron 1 of FXN indicatesFRDA. Rarely, patients will present as heterozygous for an allele havinga GAA repeat expansion and an allele having a point mutation in FXN.

FRDA Biomarkers

Frataxin protein levels. Frataxin protein levels may be measured todiagnose and monitor treatment efficacy in FRDA patients. This alsopermits multiplexing with other disease analytes and populationscreening. In this approach, frataxin protein levels may be measured bya high-throughput immunoassay. Tests can be performed employing wholeblood samples or dried blood spots to measure frataxin protein. Forwhole blood samples, frataxin levels that are ≤19 ng/mL for pediatricindividuals (less than 18 years of age) and ≤21 ng/mL for adults (18years of age or older) are consistent with a diagnosis of FRDA. Frataxinlevels that are ≥19 ng/mL for pediatric individuals and ≥21 ng/mL foradults measured using whole blood samples are not consistent with FRDA.For dried blood spot samples, frataxin levels that are ≤15 ng/mL forpediatric individuals (less than 18 years of age) and ≤21 ng/mL foradults are not consistent with FRDA. Frataxin levels that are ≥15 ng/mLfor pediatric individuals and ≥21 ng/mL for adults measured using driedblood samples are not consistent with FRDA.

High sensitive Troponin-T. High sensitive Troponin-T (hsTNT) may beuseful as a blood biomarker to indicate cumulative myocyte damageleading to fibrosis in FRDA patients (Weidemann, et al., Intl. J.Cardiol., 194:50-57 (2015)). Troponin T is a myofibrillar protein thatis present in striated musculature. There are two types of myofilaments,a thick myosin-containing filament and a thin filament consisting ofactin, tropomyosin, and troponin. Troponin is a complex of 3 proteinsubunits: troponin T, troponin I, and troponin C. Troponin T functionsto bind the troponin complex to tropomyosin.

In the cytosol, troponin T is present in soluble and protein-boundforms. The soluble or unbound pool of troponin T is released in earlystages of myocardial damage. Bound troponin T is released frommyofilaments at a later stage of irreversible myocardial damage,corresponding with degradation of myofibrils. The most common cause ofcardiac injury is myocardial ischemia (i.e., acute myocardialinfarction). Troponin T levels increase approximately 2 to 4 hours afterthe onset of myocardial necrosis, and can remain elevated for up to 14days.

Myocardial fibrosis and disease progression appear to correlate stronglywith hsTNT levels in FRDA patients. The cutoff point for the hsTNTlevels is 14 ng/L (0.014 ng/mL) (ELECSYS® Troponin T hs (TnT-hs), whichis available from Roche). Elevated serum or plasma hsTNT levels >14 ng/L(0.014 ng/mL) are seen in FRDA patients with hypertrophic cardiomyopathy(CM). Elevated hsTNT levels may indicate cumulative myocyte damageleading to fibrosis in FRDA.

II. Agents for the Modulation of Frataxin Expression

The present technology discloses an agent of the formula A-L-B, wherein-L- is a linker; A- is a Brd4 binding moiety; and -B is a nucleic acidbinding moiety.

In some embodiments, the nucleic acid binding moiety (-B) specificallybinds to a target oligonucleotide sequence. In some embodiments, thenucleic acid binding moiety (-B) specifically binds to one or morerepeats of a short oligonucleotide sequence such as a GAAoligonucleotide sequence. In some embodiments, the nucleic acid bindingmoiety (-B) is a polyamide. In some embodiments, the nucleic acidbinding moiety (-B) is a polyamide that specifically binds to one ormore repeats of an oligonucleotide sequence containing 3 to 6nucleotides, such as a GAA oligonucleotide sequence. In someembodiments, the nucleic acid binding moiety (-B) comprises anoligonucleotide sequence (e.g., containing about 15 to 30 nucleotides)that is complementary to a desired target oligonucleotide sequence. Insome embodiments, the nucleic acid binding moiety (-B) may be a nucleicacid sequence capable of hybridizing to one or more repeats of a GAAoligonucleotide sequence or to one or more repeats of a TTColigonucleotide sequence. In some embodiments, the nucleic acid bindingmoiety (-B) may be a deoxyribonucleic acid (DNA) sequence, a ribonucleicacid (RNA) sequence, or a peptide nucleic acid (PNA) sequence capable ofhybridizing to one or more repeats of a GAA oligonucleotide sequence orto one or more repeats of a TTC oligonucleotide sequence. For example,the nucleic acid binding moiety (-B) may be a deoxyribonucleic acid(DNA) sequence comprising, consisting of, or consisting essentially ofone or more repeats of a TTC sequence, including, but not limited to,TTCTTCTTC, TTCTTCTTCTTC (SEQ ID NO: 2), TTCTTCTTCTTCTTC (SEQ ID NO: 3),TTCTTCTTCTTCTTCTTC (SEQ ID NO: 4), and TTCTTCTTCTTCTTCTTCTTC (SEQ ID NO:5). In another example, the nucleic acid binding moiety (-B) may be adeoxyribonucleic acid (DNA) sequence comprising, consisting of, orconsisting essentially of one or more repeats of a GAA sequence,including, but not limited to, GAAGAAGAA, GAAGAAGAAGAA (SEQ ID NO: 6),GAAGAAGAAGAAGAA (SEQ ID NO: 7), GAAGAAGAAGAAGAAGAA (SEQ ID NO: 8), andGAAGAAGAAGAAGAAGAAGAA (SEQ ID NO: 9). In another example, the nucleicacid binding moiety (-B) may be a ribonucleic acid (RNA) sequencecomprising, consisting of, or consisting essentially of one or morerepeats of a CUU sequence, including, but not limited to, CUUCUUCUU,CUUCUUCUUCUU (SEQ ID NO: 10), CUUCUUCUUCUUCUU (SEQ ID NO: 11),CUUCUUCUUCUUCUUCUU (SEQ ID NO: 12), and CUUCUUCUUCUUCUUCUUCUU (SEQ IDNO: 13).

In some embodiments, the nucleic acid binding moiety (-B) may be adeoxyribonucleic acid (DNA) sequence comprising, consisting of, orconsisting essentially of 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, 1 to 100, 1 to 150, 1 to 200,1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 1 to 550, 1to 600, 1 to 650, 1 to 700, 1 to 750, 1 to 800, 1 to 850, 1 to 900, 1 to950, or 1 to 1000 repeats of a TTC sequence. In some embodiments, thenucleic acid binding moiety (-B) may be a DNA sequence of 5 to 10repeats of a TTC sequence (e.g., 15 to 30 nucleotide bases in length).In some embodiments, the nucleic acid binding moiety (-B) may be a DNAsequence of 5 to 6 repeats of a TTC sequence. In some embodiments, thenucleic acid binding moiety (-B) may be a DNA sequence of 5 to 7 repeatsof a TTC sequence. In some embodiments, the nucleic acid binding moiety(-B) may be a DNA sequence of 5 to 8 repeats of a TTC sequence. In someembodiments, the nucleic acid binding moiety (-B) may be a DNA sequenceof 5 to 9 repeats of a TTC sequence.

In some embodiments, the nucleic acid binding moiety (-B) may be adeoxyribonucleic acid (DNA) sequence comprising, consisting of, orconsisting essentially of 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, 1 to 100, 1 to 150, 1 to 200,1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 1 to 550, 1to 600, 1 to 650, 1 to 700, 1 to 750, 1 to 800, 1 to 850, 1 to 900, 1 to950, or 1 to 1000 repeats of a GAA sequence. In some embodiments, thenucleic acid binding moiety (-B) may be a DNA sequence of 5 to 10repeats of a GAA sequence (e.g., 15 to 30 nucleotide bases in length).In some embodiments, the nucleic acid binding moiety (-B) may be a DNAsequence of 5 to 6 repeats of a GAA sequence. In some embodiments, thenucleic acid binding moiety (-B) may be a DNA sequence of 5 to 7 repeatsof a GAA sequence. In some embodiments, the nucleic acid binding moiety(-B) may be a DNA sequence of 5 to 8 repeats of a GAA sequence. In someembodiments, the nucleic acid binding moiety (-B) may be a DNA sequenceof 5 to 9 repeats of a GAA sequence.

In some embodiments, the nucleic acid binding moiety (-B) may be aribonucleic acid (RNA) sequence comprising, consisting of, or consistingessentially of 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1to 80, 1 to 85, 1 to 90, 1 to 95, 1 to 100, 1 to 150, 1 to 200, 1 to250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 1 to 550, 1 to600, 1 to 650, 1 to 700, 1 to 750, 1 to 800, 1 to 850, 1 to 900, 1 to950, or 1 to 1000 repeats of a CUU sequence. In some embodiments, thenucleic acid binding moiety (-B) may be an RNA sequence of 5 to 10repeats of a CUU sequence (e.g., 15 to 30 nucleotide bases in length).In some embodiments, the nucleic acid binding moiety (-B) may be an RNAsequence of 5 to 6 repeats of a CUU sequence. In some embodiments, thenucleic acid binding moiety (-B) may be an RNA sequence of 5 to 7repeats of a CUU sequence. In some embodiments, the nucleic acid bindingmoiety (-B) may be an RNA sequence of 5 to 8 repeats of a CUU sequence.In some embodiments, the nucleic acid binding moiety (-B) may be an RNAsequence of 5 to 9 repeats of a CUU sequence.

In some embodiments, the nucleic acid binding moiety (-B) comprises arepeat-targeted duplex RNA, such as an anti-GAA duplex RNA thatspecifically targets GAA repeats. In some embodiments, the nucleic acidbinding moiety (-B) comprises single-stranded locked nucleic acids(LNAs), such as anti-GAA LNA oligomers that specifically target GAArepeats.

The A- subunit is typically a triazolodiazepine Brd4 binding moiety,such as a thienotriazolodiazepine Brd4 binding moiety.

The A- subunit and the -B subunit are commonly joined together by alinker -L- that has a chain having at least 10 contiguous atoms, andcommonly at least about 15 contiguous atoms in the backbone chain of thelinker. In some embodiments, the linker -L- may desirably have abackbone chain that includes no more than about 50 contiguous atoms inthe backbone of the linker, often no more than about 40 contiguousatoms, and in many instances no more than about 30 contiguous atoms inthe backbone chain of the linker. It is quite common for the linker -L-to have a backbone chain that includes about 15 to 25 contiguous atomsin the backbone of the linker.

In one aspect, A- is a triazolodiazepine Brd4 binding moiety. In someembodiments A- is a triazolodiazepine Brd4 binding moiety which may havea formula:

wherein J is N, O or CR¹¹; K is N, O or CR¹¹; with the proviso that Jand K cannot both be —O—; P is N, except when one of J or K is O, then Pis C; R¹ may be a hydrogen or optionally substituted alkyl,hydroxyalkyl, aminoalkyl, alkoxyalkyl, halogenated alkyl, hydroxyl,alkoxy, or —COOR⁴; wherein R⁴ may be a hydrogen, optionally substitutedaryl, aralkyl, cycloalkyl, heteroaryl, heteroaralkyl, heterocycloalkyl,alkyl, alkenyl, alkynyl, or cycloalkylalkyl group optionally interruptedby one or more heteroatoms; R² may be an optionally substituted aryl,alkyl, cycloalkyl, or aralkyl group; R³ may be a hydrogen, halogen, oroptionally substituted alkyl group (e.g., —(CH₂)_(b)—C(O)N(R²⁰)(R²¹),—(CH₂)_(b)—N(R²⁰)C(O)(R²¹), or halogenated alkyl group, wherein b may bean integer from 1 to 10, and R²⁰ and R²¹ may independently be a hydrogenor C₁-C₆ alkyl group (typically R²⁰ may be a hydrogen and R²¹ may be amethyl)); R¹¹ may be a hydrogen or optionally substituted alkyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group; and Ring E maybe an optionally substituted aryl or heteroaryl ring. In someembodiments, J may be N or CR¹¹. In some embodiments, P is N and J maybe CR¹¹, where R¹¹ may be —CH₃. In some embodiments, both P and J may beN.

In some embodiments A- is a Brd4 binding moiety having a formula:

wherein R¹ may be a hydrogen or an optionally substituted alkyl,hydroxyalkyl, aminoalkyl, alkoxyalkyl, halogenated alkyl, hydroxyl,alkoxy, or —COOR⁴; wherein R⁴ may be a hydrogen, or optionallysubstituted aryl, aralkyl, cycloalkyl, heteroaryl, heteroaralkyl,heterocycloalkyl, alkyl, alkenyl, alkynyl, or cycloalkylalkyl groupoptionally interrupted by one or more heteroatoms; R² may be anoptionally substituted aryl, alkyl, cycloalkyl, or aralkyl group; R³ maybe a hydrogen, halogen, or optionally substituted alkyl group (e.g.,—(CH₂)_(x)—C(O)N(R²⁰)(R²¹), —(CH₂)—N(R²⁰)C(O)(R²¹), or halogenated alkylgroup, wherein x may be an integer from 1 to 10, and R²⁰ and R²¹ mayindependently be a hydrogen or C₁-C₆ alkyl group (typically R²⁰ may be ahydrogen and R²¹ may be a methyl)); and Ring E may be an optionallysubstituted aryl or heteroaryl group. In some embodiments, x may be aninteger from 1 to 6. In some embodiments, x may be an integer from 1 to3.

In some embodiments, A- is a Brd4 binding moiety having a formula:

wherein x, R¹, R², R³, and Ring E are as defined herein.

In some embodiments, A- is a thienotriazolodiazepine Brd4 bindingmoiety. In some embodiments A- is a thienotriazolodiazepine Brd4 bindingmoiety which may have a formula:

wherein R² may be an aryl group optionally substituted with halogen,—OR⁶, —SR⁶, —N(R⁶)₂, —N(R⁶)COR⁹, or one or more optionally substitutedalkyl, alkenyl, alkynyl, aryl, heteroaryl, amino, or amido groups,wherein R⁶ and R⁹ may independently be a hydrogen or alkyl group; R¹ andR³ may independently be a hydrogen or optionally substituted alkylgroup; and R⁵ and R⁷ may independently be a hydrogen, alkyl, alkenyl,alkynyl, halogen, —OH, —SH, or —NH₂. In some embodiments, R² may be aphenyl group optionally substituted with one or more alkyl, cyano,halogenated alkyl, alkoxy, hydroxyalkyl, and/or halogen substituents. Insome embodiments, R² may be a phenyl group optionally substituted withone or more halogenated alkyl groups. In some embodiments, R² may be aphenyl group optionally substituted with one or more halogens. In someembodiments, R² may be a phenyl group substituted with one, two, three,four or five halogens.

In some embodiments, A- is a thienotriazolodiazepine Brd4 binding moietyhaving a formula:

wherein x, R¹, R², R³, R⁵, and R⁷ are as defined herein.

In some embodiments A- is a Brd4 binding moiety having a formula:

wherein R³ may be a hydrogen or optionally substituted C₁-C₆ alkylgroup; R¹, R⁵, and R⁷ are each independently hydrogen, methyl, ethyl, orhalomethyl (e.g., trifluoromethyl); and R⁸ may be a halogen, optionallysubstituted aryl, amino, or amido group. In some embodiments, R³ may bea hydrogen or —CH₃. In some embodiments, R³ may be —CH₃. In someembodiments, R¹, R⁵, and R⁷ are each independently hydrogen, methyl,ethyl, or trifluoromethyl. In some embodiments, R¹, R⁵, and R⁷ are eachmethyl or ethyl. In some embodiments, R¹, R⁵, and R⁷ are each ethyl. Insome embodiments, R¹, R⁵, and R⁷ are each methyl. In some embodiments,R¹, R⁵, and R⁷ are each independently hydrogen. In some embodiments, R¹,R⁵, and R⁷ are each independently trifluoromethyl. In some embodiments,R³ may be hydrogen or —CH₃; R¹, R⁵, and R⁷ may be methyl; and R⁸ may bechloro. In some embodiments, R³ may be hydrogen; R¹, R⁵, and R⁷ may bemethyl; and R⁸ may be chloro. In some embodiments, R¹ may be a hydrogen,methyl, ethyl, or halomethyl. In some embodiments, R¹ may be atrifluoromethyl.

In some embodiments, R⁸ may be a halogen. In some embodiments, R⁸ may be—Cl. In some embodiments, R⁸ may be —F. In some embodiments, R⁸ may be aphenyl group optionally substituted with one or more cyano and/or alkoxygroups. In some embodiments, R⁸ may be a phenyl group substituted with acyano group. In some embodiments, R⁸ may be a phenyl group substitutedwith a methoxy group. In some embodiments, R⁸ may be an optionallysubstituted amino group. In some embodiments, R⁸ may be an amino groupsubstituted with an optionally substituted phenyl, benzyl, or heteroarylgroup and/or alkyl group. In some embodiments, R⁸ may be an amino groupsubstituted with a phenyl group. In some embodiments, R⁸ may be an aminogroup substituted with a halogenated phenyl group. In some embodiments,R⁸ may be an amino group substituted with a methyl and a halogenatedphenyl group. In some embodiments, R⁸ may be an amino group substitutedwith a heteroaryl group. In some embodiments, R⁸ may be an amino groupsubstituted with a pyridyl group. In some embodiments, R⁸ may be anamino group substituted with a benzyl group. In some embodiments, R⁸ maybe an amido group substituted with an alkyl, aralkyl, or alkaryl group.In some embodiments, R⁸ may be an amido group substituted with anaralkyl group. In some embodiments, R⁸ may be an amido group substitutedwith —(CH₂)_(r)-phenyl group, wherein t is an integer from 1 to 10. Insome embodiments, t may be 1 or 2.

In some embodiments A- is a Brd4 binding moiety having a formula:

wherein x, R¹, R³, R⁵, R⁷, and R⁸ are as defined herein.

In some embodiments A- is a Brd4 binding moiety having a formula:

wherein x is as defined herein.

In some embodiments, -B may comprise one or more of the followingsubunits:

in which Z is typically hydrogen, amino, or an amido group.

In some embodiments, the one or more repeats may be GAA. In someembodiments, -B may specifically bind to a one or more repeats of a GAAoligonucleotide sequence. In some embodiments, -B may include-X-(β-Py-Im)_(n)-β-Py-TRM; where X is -β-Im-, -β-Py-, -β-, or a bond; nis 1-10; and -TRM is -ImT or -CTh; with the proviso that one of the-β-Py-Im- trimers may be replaced by a -β-Im-Im- trimer. In someembodiments, -B may include -X-(β-Py-Im)_(n)- (β-Py-ImT); wherein: X is-β-Im-, -β-Py-, -β-, or a bond; Z is hydrogen, amino, or amido group;and n is 0 to 10; with the proviso that when n is at least 1, one of the-β-Py-Im- trimers may be replaced by a -β-Im-Im- trimer.

In some embodiments, Z may be —NR^(B)R^(B) or -N+R^(A)R^(B)R^(B);wherein R^(A) may be hydrogen; and R^(B) may be a hydrogen, C₁-C₆ alkyl,C₁-C₆ alkenyl, or C₁-C₆ alkynyl group. In some embodiments, Z may be—N(R^(A))C(O)R^(B); wherein R^(A) may be hydrogen; and R^(B) may be ahydrogen, C₁-C₆ alkyl, C₁-C₆ alkenyl, or C₁-C₆ alkynyl group. In someembodiments, R^(B) may be hydrogen or C₁-C₆ alkyl group. In someembodiments, R^(B) may be hydrogen or —CH₃. In certain embodiments, Zmay be —NH₂. In certain embodiments, Z may be —NH₃ ⁺. In certainembodiments, Z may be hydrogen.

In some embodiments, n may be an integer from 1 to 10. In someembodiments, n may be 1, 2, 3, 4, or 5. In certain embodiments, n may be1 or 2. In some embodiments, n may be 1 or 2 and none of the -β-Py-Im-trimers are replaced by a -β-Im-Im- trimer. In some embodiments, n maybe 1 or 2 and one of the -β-Py-Im- trimers is replaced by a-β-Im-Im-trimer.

In some embodiments, -B may be -(β-Py-Im)_(n)-(β-Py-ImT); wherein Z maybe hydrogen or NH₃ ⁺ and n may be 1 or 2.

In some embodiments, -B may include -β-Im-β-Py-Im-β-Py-ImT,-β-β-Py-Im-β-Py-ImT, -β-Py-Im-β-Py-ImT, -β-Im-β-Py-Im-β-Py-Im-β-Py-ImT,-β-Py-Im-β-Py-Im-β-Py-ImT, and/or -β-β-Py-Im-β-Py-Im-β-Py-ImT; in whichZ may be hydrogen. In some embodiments, -B may include-β-Im-β-Py-Im-β-Py-Im-β-Py-ImT, -β-Im-β-Py-Im-β-Im-Im-β-Py-ImT, and/or-β-Im-β-Im-Im-β-Py-Im-β-Py-ImT; in which Z may be hydrogen. In someembodiments, -B may include -β-Py-β-Py-Im-β-Py-ImT, -β-β-Py-Im-β-Py-ImT,-β-Im-β-Py-Im-β-Py-Im-β-Py-ImT, -β-Py-β-Py-Im-β-Py-Im-β-Py-ImT,-β-Py-Im-β-Py-Im-β-Py-ImT, -β-Py-β-Py-Im-β-Py-CTh and/or-β-β-Py-Im-β-Py-Im-β-Py-ImT, -β-Py-Im-β-Py-CTh.

In some embodiments -L- may be a covalent linking group. In someembodiments -L- may be a linker having a contiguous backbone chain whichincludes at least about 10 atoms. In some embodiments, -L- may have acontiguous backbone chain that includes about 15 to 250 atoms. In someembodiments, -L- may be a combination of one or more optionallysubstituted arylene, aralkylene, cycloalkylene, heteroarylene,heteroaralkylene, heterocycloalkylene, alkylene, alkenylene, alkynylene,or cycloalkylalkylene, optionally interrupted by one or moreheteroatoms, amido, or carboxyl groups. In some embodiments, -L- mayinclude a combination of one or more linking moieties selected from thegroup consisting of —O—, —(CH₂)—, —(CH₂CH₂O)_(y)—,—(OCH₂CH₂)_(y)—C(O)NR′—, —NR′C(O)—, —C(O)—, —NR*—, and

wherein R′ and R* are each independently a hydrogen or C₁-C₆ alkyl; andx and y are each independently an integer from 1 to 10. In someembodiments, R′ may be a hydrogen and R* may be —CH₃.

In some embodiments, -L- may include—(CH₂)_(x)—C(O)N(R′)—(CH₂)_(Q)—N(R*)—(CH₂)_(Q)—N(R′)C(O)—(CH₂)—C(O)N(R′)—,—(CH₂)—C(O)N(R′)—(CH₂CH₂O), —(CH₂)_(x)—C(O)N(R′)—,—C(O)N(R′)—(CH₂)_(Q)—N(R*)—(CH₂)_(Q)—N(R′)C(O)—(CH₂)—,—(CH₂)—O—(CH₂CH₂O)_(y)—(CH₂)_(x)—N(R′)C(O)—(CH₂)—, or—N(R′)C(O)—(CH₂)_(x)—C(O)N(R′)—(CH₂)—O—(CH₂CH₂O)_(y)—(CH₂)_(x)—, whereinR* may be methyl, R′ may be hydrogen, Q may be an integer from 2 to 10,and x and y may independently be an integer from 1 to 10. In someembodiments, R′ may be a hydrogen; R* may be —CH₃; x and y mayindependently be an integer from 1 to 3; and Q may be 2 or 3.

In some embodiments, -L- may include one or more linking moietiesselected from (Gly-Ser-Gly)_(V) (SEQ ID NO: 14) and (Gly-Gly-Ser)_(W)(SEQ ID NO: 15), where v and w are typically an integer from 1 to about10.

In one aspect, the present technology discloses an agent having aformula A-L-B, wherein L is a linker having a backbone chain which mayinclude at least 10 atoms; -B is a polyamide that specifically binds toone or more repeats of a GAA oligonucleotide sequence; and A- is a Brd4binding moiety, such as a triazolodiazepine Brd4 binding moiety having astructure:

wherein R³ and R¹⁰ may independently be a hydrogen, halogen, oroptionally substituted alkyl group (e.g., —(CH₂)_(x)—C(O)N(R²⁰)(R²¹),—(CH₂)_(x)—N(R²⁰)C(O)(R²¹), or halogenated alkyl group, wherein R²⁰ andR²¹ may independently be a hydrogen or C₁-C₆ alkyl group (typically R²⁰may be a hydrogen and R²¹ may be a methyl), and x is as defined herein);R¹ may be a hydrogen or optionally substituted alkyl, hydroxyl, alkoxy,or —COOR⁴; wherein R⁴ may be a hydrogen, optionally substituted aryl,aralkyl, cycloalkyl, heteroaryl, heteroaralkyl, heterocycloalkyl, alkyl,alkenyl, alkynyl, or cycloalkylalkyl group optionally interrupted by oneor more heteroatoms; and Ring E may be an optionally substituted aryl orheteroaryl group. In some embodiments, R¹ may be a hydroxyalkyl,aminoalkyl, alkoxyalkyl, or halogenated alkyl group. In someembodiments, R¹ may be a hydrogen, methyl, ethyl, or halomethyl. In someembodiments, R¹ may be a trifluoromethyl.

In some embodiments, R³ and R¹⁰ may independently be a hydrogen oroptionally substituted alkyl. In some embodiments, R³ and R¹⁰ mayindependently be hydrogen or optionally substituted C₁-C₆ alkyl group.In some embodiments, R³ and R¹⁰ may independently be a—(CH₂)_(x)—C(O)N(R′)R″, wherein R′ and R″ are each independently ahydrogen or C₁-C₆ alkyl and x is an integer from 1 to 10. In someembodiments, R′ is a hydrogen. In some embodiment R″ is a —CH₃. In someembodiments, A- is a thienotriazolodiazepine Brd4 binding moiety. Insome embodiment, -L- is a linker as defined herein and -B is a polyamideas defined herein

III. Methods for the Modulation of Frataxin Expression

The present technology relates to methods and compositions formodulating frataxin (FXN) gene expression. In particular, the methodsand compositions relate to the use of one or more agents to stimulateFXN transcription.

In one aspect, the methods and compositions of the present technologyrelate to agents having a formula A-L-B, wherein -L- is a linker; A- isa Brd4 binding moiety; and -B is a nucleic acid binding moiety and theuse of one or more of these agents to stimulate FXN transcription. Insome embodiments, the nucleic acid binding moiety (-B) specificallybinds to a target oligonucleotide sequence. In some embodiments, thenucleic acid binding moiety (-B) specifically binds to one or morerepeats of a short oligonucleotide sequence such as a GAAoligonucleotide sequence. In some embodiments, the nucleic acid bindingmoiety (-B) is a polyamide. In some embodiments, the nucleic acidbinding moiety (-B) is a polyamide that specifically binds to one ormore repeats of an oligonucleotide sequence containing 3 to 6nucleotides, such as a GAA oligonucleotide sequence. In someembodiments, the nucleic acid binding moiety (-B) comprises anoligonucleotide sequence (e.g., containing about 15 to 30 nucleotides)that is complementary to a desired target oligonucleotide sequence. Insome embodiments, the nucleic acid binding moiety (-B) may be a nucleicacid sequence capable of hybridizing to one or more repeats of a GAAoligonucleotide sequence or to one or more repeats of a TTColigonucleotide sequence. In some embodiments, the nucleic acid bindingmoiety (-B) may be a deoxyribonucleic acid (DNA) sequence, a ribonucleicacid (RNA) sequence, or a peptide nucleic acid (PNA) sequence capable ofhybridizing to one or more repeats of a GAA oligonucleotide sequence orto one or more repeats of a TTC oligonucleotide sequence. For example,the nucleic acid binding moiety (-B) may be a deoxyribonucleic acid(DNA) sequence comprising, consisting of, or consisting essentially ofone or more repeats of a TTC sequence, including, but not limited to,TTCTTCTTC, TTCTTCTTCTTC (SEQ ID NO: 2), TTCTTCTTCTTCTTC (SEQ ID NO: 3),TTCTTCTTCTTCTTCTTC (SEQ ID NO: 4), and TTCTTCTTCTTCTTCTTCTTC (SEQ ID NO:5). In another example, the nucleic acid binding moiety (-B) may be adeoxyribonucleic acid (DNA) sequence comprising, consisting of, orconsisting essentially of one or more repeats of a GAA sequence,including, but not limited to, GAAGAAGAA, GAAGAAGAAGAA (SEQ ID NO: 6),GAAGAAGAAGAAGAA (SEQ ID NO: 7), GAAGAAGAAGAAGAAGAA (SEQ ID NO: 8), andGAAGAAGAAGAAGAAGAAGAA (SEQ ID NO: 9). In another example, the nucleicacid binding moiety (-B) may be a ribonucleic acid (RNA) sequencecomprising, consisting of, or consisting essentially of one or morerepeats of a CUU sequence, including, but not limited to, CUUCUUCUU,CUUCUUCUUCUU (SEQ ID NO: 10), CUUCUUCUUCUUCUU (SEQ ID NO: 11),CUUCUUCUUCUUCUUCUU (SEQ ID NO: 12), and CUUCUUCUUCUUCUUCUUCUU (SEQ IDNO: 13).

In some embodiments, the nucleic acid binding moiety (-B) may be adeoxyribonucleic acid (DNA) sequence comprising, consisting of, orconsisting essentially of 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, 1 to 100, 1 to 150, 1 to 200,1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 1 to 550, 1to 600, 1 to 650, 1 to 700, 1 to 750, 1 to 800, 1 to 850, 1 to 900, 1 to950, or 1 to 1000 repeats of a TTC sequence. In some embodiments, thenucleic acid binding moiety (-B) may be a DNA sequence of 5 to 10repeats of a TTC sequence (e.g., 15 to 30 base pairs in length). In someembodiments, the nucleic acid binding moiety (-B) may be a DNA sequenceof 5 to 10 repeats of a TTC sequence (e.g., 15 to 30 nucleotide bases inlength). In some embodiments, the nucleic acid binding moiety (-B) maybe a DNA sequence of 5 to 6 repeats of a TTC sequence. In someembodiments, the nucleic acid binding moiety (-B) may be a DNA sequenceof 5 to 7 repeats of a TTC sequence. In some embodiments, the nucleicacid binding moiety (-B) may be a DNA sequence of 5 to 8 repeats of aTTC sequence. In some embodiments, the nucleic acid binding moiety (-B)may be a DNA sequence of 5 to 9 repeats of a TTC sequence.

In some embodiments, the nucleic acid binding moiety (-B) may be adeoxyribonucleic acid (DNA) sequence comprising, consisting of, orconsisting essentially of 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, 1 to 100, 1 to 150, 1 to 200,1 to 250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 1 to 550, 1to 600, 1 to 650, 1 to 700, 1 to 750, 1 to 800, 1 to 850, 1 to 900, 1 to950, or 1 to 1000 repeats of a GAA sequence. In some embodiments, thenucleic acid binding moiety (-B) may be a DNA sequence of 5 to 10repeats of a GAA sequence (e.g., 15 to 30 base pairs in length). In someembodiments, the nucleic acid binding moiety (-B) may be a DNA sequenceof 5 to 6 repeats of a GAA sequence. In some embodiments, the nucleicacid binding moiety (-B) may be a DNA sequence of 5 to 7 repeats of aGAA sequence. In some embodiments, the nucleic acid binding moiety (-B)may be a DNA sequence of 5 to 8 repeats of a GAA sequence. In someembodiments, the nucleic acid binding moiety (-B) may be a DNA sequenceof 5 to 9 repeats of a GAA sequence.

In some embodiments, the nucleic acid binding moiety (-B) may be aribonucleic acid (RNA) sequence comprising, consisting of, or consistingessentially of 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 35, 1to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1to 80, 1 to 85, 1 to 90, 1 to 95, 1 to 100, 1 to 150, 1 to 200, 1 to250, 1 to 300, 1 to 350, 1 to 400, 1 to 450, 1 to 500, 1 to 550, 1 to600, 1 to 650, 1 to 700, 1 to 750, 1 to 800, 1 to 850, 1 to 900, 1 to950, or 1 to 1000 repeats of a CUU sequence. In some embodiments, thenucleic acid binding moiety (-B) may be an RNA sequence of 5 to 10repeats of a CUU sequence (e.g., 15 to 30 nucleotide bases in length).In some embodiments, the nucleic acid binding moiety (-B) may be an RNAsequence of 5 to 6 repeats of a CUU sequence. In some embodiments, thenucleic acid binding moiety (-B) may be an RNA sequence of 5 to 7repeats of a CUU sequence. In some embodiments, the nucleic acid bindingmoiety (-B) may be an RNA sequence of 5 to 8 repeats of a CUU sequence.In some embodiments, the nucleic acid binding moiety (-B) may be an RNAsequence of 5 to 9 repeats of a CUU sequence.

In some embodiments, the nucleic acid binding moiety (-B) comprises arepeat-targeted duplex RNA, such as an anti-GAA duplex RNA thatspecifically targets GAA repeats. In some embodiments, the nucleic acidbinding moiety (-B) comprises single-stranded locked nucleic acids(LNAs), such as anti-GAA LNA oligomers that specifically target GAArepeats.

In some embodiments, the present technology relates to methods andcompositions for preventing or treating Friedreich's ataxia in a subjectin need thereof. In some embodiments, the methods and compositions ofthe present technology increase the level of frataxin (FXN) mRNA levelsin a cell. In some embodiments, the methods and compositions of thepresent technology increase frataxin protein levels in a cell. In someembodiments, the methods and compositions of the present technologyprevent one or more signs or symptoms of Friedreich's ataxia in asubject. In some embodiments, the methods and compositions of thepresent technology reduce the likelihood that a subject with riskfactors for Friedreich's ataxia will develop one or more signs orsymptoms of Friedreich's ataxia, or will delay the onset of Friedreich'sataxia.

As described in more detail below, the agents disclosed herein combine anucleic acid binding moiety, such as a DNA-binding polyamide subunit(-B) that specifically binds to one or more repeats of a GAAoligonucleotide sequence, with a linker (-L-) and a Brd4 binding moiety(A-). In some embodiments, the Brd4 binding moiety has a structurerelated to a bromodomain inhibitor (e.g., JQ1). Though not wishing to bebound by any particular theory, as depicted by the model transcriptionelongation stimulator in FIG. 12, it is believed that the polyamide-JQ1conjugate binds selectively to one or more repeats in an oligonucleotidesequence (e.g., one or more GAA trinucleotide repeats), and through theinteraction of JQ1 with Brd4, recruits the super elongation complex(SEC) to stimulate and restart the paused RNA polymerase-II (Pol II)transcription complex, thereby resulting in transcription of frataxin(FXN) mRNA.

JQ1 is one example of a selective small-molecule bromodomain inhibitor.Specifically, JQ1 is a thieno-triazolo-1,4-diazepine that displacesbromodomain and extra-terminal (BET) family members (e.g., Brd4) fromchromatin by competitively binding to the acetyl-lysine recognitionpocket. (Delmore, et al., Cell, 146(6):904-917 (2011)). Delmore, et al.have shown that Brd4 is strongly enriched at immunoglobulin heavy chain(IgH) enhancers in MM cells bearing IgH rearrangement at the MYC locusand that JQ1 depletes enhancer-bound Brd4 and inhibits MYC transcriptionin a dose- and time-dependent manner. In addition, JQ1 has been shown tobind and displace BRD4 from chromatin thereby inhibiting transcriptionof Brd4-dependent genes. (See Filippakopoulos, et al. Nature 468:1067-1073). Accordingly, previous studies have established a role forJQ1 and related structures in binding Brd4 to inhibit transcription.FIG. 13 depicts a crystal structure showing (S)-JQ1 bound to Brd4(Protein Data Bank accession 3MXF). The tert-butyl group, highlighted byan asterisk, projects out of the binding pocket of Brd4, indicating thatchemical substitution at this position would likely be tolerated.

Examples of compounds (including controls) tested by the methods of thepresent technology to determine their ability to function astranscription elongation stimulators include Polyamide 1 (“1”) ControlConjugate 2 (“2”), Polyamide 3 (“3”) and Agent 4 (“4”), as depicted inFIG. 14.

Brd4 has been reported to play a role in mediating transcriptionalelongation, functioning to recruit the positive transcription elongationfactor (P-TEFb), which plays an essential role in the regulation oftranscription by RNA polymerase-II (Pol II). In humans, there aremultiple forms of P-TEFb, which contain the catalytic subunit, Cdk9, andtogether with several other cyclin subunits associate with Brd4 to forma complex of proteins called the super elongation complex (SEC). Withoutwishing to be bound by theory, it is believed that recruitment of thiscomplex facilitates the transition of promoter-proximal paused PolIIinto productive elongation.

Engineered polyamides have been shown to bind DNA with high affinity.For example, pyrrole-imidazole polyamides are cell-permeable smallmolecules that can be designed to bind a variety of DNA sequences.(Burnett et al. PNAS 103(31):11497-11502). Previous studies havereported that DNA sequence-specific polyamides that bind GAAtrinucleotide repeats lead to an approximate 3-fold increase intranscription of frataxin in cell culture when provided at aconcentration of 2 μM for 7 days (polyamide replenished on days 3 and5). However, no significant changes in frataxin mRNA levels wereobserved with shorter incubation times. (Burnett et al.).

By contrast, the methods disclosed herein allow for the activation ofFXN in a Friedreich's ataxia cell line with as little as a 100 nMconcentration of the agents of the present technology after only 24hours. For example, as shown in FIG. 1A, the agents of the presenttechnology, which comprise JQ1, a general transcription inhibitor,potently activate FXN to produce a greater than 4-fold increase in mRNAlevels when administered to FRDA cells at a concentration of 500 nMafter only 24 hours. This approximate 4-fold increase returns mRNAlevels to those typically present in asymptomatic heterozygotes. Also,as shown in FIG. 1A, the agents of the present technology activate FXNto produce a greater than 8-fold increase in mRNA levels whenadministered to FRDA cells at a concentration of 1 μM after only 24hours, thereby returning FXN mRNA to near-wild-type levels (See FIG.1C). The ability to activate FXN mRNA production with relatively lowconcentrations of the agents of the present technology may beparticularly advantageous in the clinical setting.

The methods disclosed herein can be used to stimulate FXN mRNAtranscription by culturing the cells under cell-type specific conditionsknown in the art and contacting cells with an effective amount of one ormore of the agents of the present technology according to any methodknown to those in the art for contacting a cell. In some embodiments ofthe methods disclosed herein, dorsal root ganglion neurons are used. Insome embodiments of the methods disclosed herein, cardiomyocytes areused. In some embodiments of the methods disclosed herein, pancreaticbeta cells are used. In some embodiments of the methods disclosedherein, peripheral blood mononuclear cells (PBMCs) are used. In someembodiments of the methods disclosed herein, B-lymphocytes are used. Insome embodiments of the methods disclosed herein, lymphoblastoid celllines are used. In some embodiments of the methods disclosed herein,fibroblasts are used. In some embodiments, the cells are derived from apatient subject.

In some embodiments, the agent is provided at a level sufficient to bindat least 2, 3, or more repeats of the oligonucleotide sequence. In someembodiments, of the methods disclosed herein, the cells are contactedwith one or more agents of the present technology at a concentration ofabout 10 nM. In some embodiments, the cells are contacted with one ormore agents at a concentration of about 50 nM. In some embodiments, thecells are contacted with one or more agents at a concentration of about100 nM. In some embodiments, the cells are contacted with one or moreagents at a concentration of about 200 nM. In some embodiments, thecells are contacted with one or more agents at a concentration of about300 nM. In some embodiments, the cells are contacted with one or moreagents at a concentration of about 400 nM. In some embodiments, thecells are contacted with one or more agents at a concentration of about500 nM. In some embodiments, the cells are contacted with one or moreagents at a concentration of about 600 nM. In some embodiments, thecells are contacted with one or more agents at a concentration of about700 nM. In some embodiments, the cells are contacted with one or moreagents at a concentration of about 800 nM. In some embodiments, thecells are contacted with one or more agents at a concentration of about900 nM. In some embodiments, the cells are contacted with one or moreagents at a concentration of about 1 μM. In some embodiments, the cellsare contacted with one or more agents at a concentration of about 2 μM.In some embodiments, the cells are contacted with one or more agents ata concentration of about 3 μM. In some embodiments, the cells arecontacted with one or more agents at a concentration of about 4 μM. Insome embodiments, the cells are contacted with one or more agents at aconcentration of about 5 μM.

In some embodiments, of the methods disclosed herein, the cells areharvested for subsequent measurement of mRNA and/or protein levels atabout 6 hours after having been contacted with one or more doses of theagents of the present technology. In some embodiments, of the methodsdisclosed herein, the cells are harvested for subsequent measurement ofmRNA and/or protein levels at about 12 hours after having been contactedwith one or more doses of the agents of the present technology. In someembodiments, of the methods disclosed herein, the cells are harvestedfor subsequent measurement of frataxin mRNA and/or protein levels atabout 24 hours after having been contacted with one or more doses of theagents of the present technology. In some embodiments, the cells areharvested at about 2 days after having been contacted with one or moredoses of the agents of the present technology. In some embodiments, thecells are harvested at about 3 days after having been contacted with oneor more doses of the agents of the present technology. In someembodiments, the cells are harvested at about 4 days after having beencontacted with one or more doses of the agents of the presenttechnology. In some embodiments, the cells are harvested at about 5 daysor more after having been contacted with one or more doses of the agentsof the present technology.

In some embodiments, the present disclosure provides a method formodulating transcription of a gene that includes multiple repeats of anoligonucleotide sequence containing 3 to 6 nucleotides, such as a GAAoligonucleotide repeat expansion. Without wishing to be bound by theory,the modulation of transcription is effected by contacting the gene withan agent of the present technology having a formula A-L-B, wherein -L-is a linker; A- is a Brd4 binding moiety; and -B is a polyamide thatspecifically binds to one or more repeats of the oligonucleotidesequence, thereby modulating the transcription of the gene. In someembodiments, the gene is a frataxin (FXN) gene. In some embodiments, thenumber of repeats in the oligonucleotide expansion is greater than 50,greater than 70, greater than 100, or in a range of 66-1700.

In some embodiments, the present disclosure provides a method forincreasing frataxin (FXN) mRNA levels in a cell comprising contactingthe cell with an effective amount of any one or more of the agentsdescribed in Section II.

In some embodiments, the present disclosure provides a method forincreasing frataxin (FXN) protein levels in a cell comprising contactingthe cell with an effective amount of any one or more of the agentsdescribed in Section II.

In some embodiments, the present disclosure provides a method fortreating a genetic condition associated with a gene comprising aplurality of repeats of a GAA oligonucleotide sequence, comprisingadministering to the subject a therapeutically effective amount of anyone or more of the agents described in Section II. In some embodiments,the disease is Friedreich's ataxia (FRDA).

In some embodiments of the present method, the cell may include a fusiongene including at least about 30 GAA repeats, a sequence encoding afunctional frataxin polypeptide sequence connected to a heterologouspolypeptide sequence (e.g., a full length frataxin (FXN) gene ornucleotide sequence encoding a functional frataxin polypeptide fused tothe heterologous polypeptide sequence). The cell may include a reportergene fused to the 3′-end of the frataxin (FXN) gene or to the 3′-end ofa sequence encoding a functional frataxin polypeptide sequence. As usedherein, the term “functional frataxin polypeptide” refers topolypeptides including at least amino acid residues 81-210 of humanfrataxin. In some embodiments, the reporter gene comprises aluminescence-based reporter gene. In some embodiments theluminescence-based reporter gene is a luciferase reporter gene, e.g., agene encoding firefly luciferase, Renilla luciferase, or the like. Inother embodiments, the reporter gene may be a gene for a selectablemarker, e.g., an antibiotic resistance gene.

IV. Modes of Administration and Pharmaceutical Compositions

Any method known to those in the art for contacting a cell, organ, ortissue with compositions such as the agents of the present technology,or pharmaceutically acceptable salts thereof, may be employed. Suitablemethods include in vitro, ex vivo, or in vivo methods.

In vitro methods typically include cultured samples. For example, a cellcan be placed in a reservoir (e.g., tissue culture plate), and incubatedwith an agent under appropriate conditions suitable for obtaining thedesired result. Suitable incubation conditions can be readily determinedby those skilled in the art.

Ex vivo methods typically include cells, organs, or tissues removed froma mammal, such as a human. The cells, organs or tissues can, forexample, be incubated with the agent under appropriate conditions. Thecontacted cells, organs, or tissues are typically returned to the donor,placed in a recipient, or stored for future use. Thus, the compound isgenerally in a pharmaceutically acceptable carrier.

In vivo methods typically include the administration of an agent such asthose described herein, to a mammal such as a human. When used in vivofor therapy, an agent of the present technology is administered to amammal in an amount effective in obtaining the desired result ortreating the mammal.

An effective amount of an agent of the present technology useful in thepresent methods, such as in a pharmaceutical composition or medicament,may be administered to a mammal in need thereof by any of a number ofwell-known methods for administering pharmaceutical compositions ormedicaments. The agents of the present technology may be administeredsystemically or locally.

The agents of the present technology may be formulated as apharmaceutically acceptable salt. The term “pharmaceutically acceptablesalt” means a salt prepared from a base or an acid which is acceptablefor administration to a patient, such as a mammal (e.g., salts havingacceptable mammalian safety for a given dosage regimen).

The agents of the present technology described herein can beincorporated into pharmaceutical compositions for administration, singlyor in combination, to a subject for the treatment or prevention ofFreidreich's ataxia. Such compositions typically include the activeagent and a pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” includes saline, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith the intended route of administration. Routes of administrationinclude, for example, parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, respiratory (e.g., inhalation),transdermal (topical), and transmucosal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates, and agents for the adjustment of tonicity, suchas sodium chloride or dextrose. The pH can be adjusted with acids orbases, such as hydrochloric acid or sodium hydroxide. The preparationcan be enclosed in ampoules, disposable syringes or multiple-dose vialsmade of glass or plastic. For convenience of the patient or treatingphysician, the dosing formulation can be provided in a kit containingall necessary equipment (e.g., vials of drug, vials of diluent, syringesand needles) for a course of treatment (e.g., 7 days of treatment).

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL® (BASF, Parsippany, N.J., USA) or phosphate buffered saline (PBS). Inall cases, a composition for parenteral administration must be sterileand should be formulated for ease of syringeability. The compositionshould be stable under the conditions of manufacture and storage, andmust be shielded from contamination by microorganisms such as bacteriaand fungi.

The pharmaceutical compositions can include a carrier, which can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thiomerasol, and the like. Glutathione and other antioxidants can beincluded to prevent oxidation. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In one embodiment, agent of the present technology is administeredintravenously. For example, an agent of the present technology may beadministered via rapid intravenous bolus injection. In some embodiments,the agent of the present technology is administered as a constant-rateintravenous infusion.

The agent of the present technology may also be administered orally,topically, intranasally, intramuscularly, subcutaneously, ortransdermally. In one embodiment, transdermal administration is byiontophoresis, in which the charged composition is delivered across theskin by an electric current.

Other routes of administration include intracerebroventricularly orintrathecally. Intracerebroventricularly refers to administration intothe ventricular system of the brain. Intrathecally refers toadministration into the space under the arachnoid membrane of the spinalcord. Thus, in some embodiments, intracerebroventricular or intrathecaladministration is used for those diseases and conditions which affectthe organs or tissues of the central nervous system.

For systemic, intracerebroventricular, intrathecal, topical, intranasal,subcutaneous, or transdermal administration, formulations of the agentsof the present technology may utilize conventional diluents, carriers,or excipients etc., such as those known in the art to deliver the agentsof the present technology. For example, the formulations may compriseone or more of the following: a stabilizer, a surfactant, such as anonionic surfactant, and optionally a salt and/or a buffering agent. Theagents of the present technology may be delivered in the form of anaqueous solution, or in a lyophilized form.

V. Dosage

The dosage ranges described herein are exemplary and are not intended tobe limiting.

Dosage, toxicity, and therapeutic efficacy of the agents of the presenttechnology can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any agent of thepresent technology used in the methods described herein, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

Typically, an effective amount of the agent of the present technology,sufficient for achieving a therapeutic or prophylactic effect, rangesfrom about 0.000001 mg per kilogram body weight per day to about 10,000mg per kilogram body weight per day. In some embodiments, the dosageranges will be from about 0.0001 mg per kilogram body weight per day toabout 100 mg per kilogram body weight per day. For example dosages canbe 1 mg/kg body weight or 10 mg/kg body weight every day, every two daysor every three days or within the range of 1-10 mg/kg every week, everytwo weeks or every three weeks. In one embodiment, a single dosage ofthe agent of the present technology ranges from 0.1-10,000 microgramsper kg body weight. An exemplary treatment regimen entailsadministration once per day or once a week. Intervals can also beirregular as indicated by measuring blood levels of glucose or insulinin the subject and adjusting dosage or administration accordingly. Insome methods, dosage is adjusted to achieve a desired fasting glucose orfasting insulin concentration. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, or until thesubject shows partial or complete amelioration of symptoms of disease.Thereafter, the patient can be administered a prophylactic regimen.

In some embodiments, a therapeutically effective amount of the agent ofthe present technology is defined as a concentration of the agent of thepresent technology at the target tissue of 10⁻¹¹ to 10⁻⁶ molar, e.g.,approximately 10⁻⁷ molar. This concentration may be delivered bysystemic doses of 0.01 to 100 mg/kg or equivalent dose by body surfacearea. The schedule of doses is optimized to maintain the therapeuticconcentration at the target tissue, such as by single daily or weeklyadministration, but also including continuous administration (e.g.,parenteral infusion or transdermal application).

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and thepresence of other diseases. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

VI. Therapeutic Applications

The methods and compositions described herein have a variety ofapplications and therapeutic uses. In some embodiments, the methods andcompositions disclosed herein are directed to the use of any one or moreof the agents described in Section II to stimulate FXN transcription. Insome embodiments, the present technology relates to methods andcompositions for preventing or treating Friedreich's ataxia in a subjectin need thereof. In some embodiments, the methods and compositions ofthe present technology increase the level of frataxin (FXV) mRNA levelsin a cell. In some embodiments, the methods and compositions of thepresent technology increase frataxin protein levels in a cell. In someembodiments, the methods and compositions of the present technologyprevent or treat one or more signs or symptoms of Friedreich's ataxiaincluding, but not limited to, e.g., muscle weakness, loss ofcoordination, lack of reflexes in lower limbs, loss of ability to feelvibrations in lower limbs, vision impairment, color vision changes,involuntary eye movements, pes cavus, hearing impairment, slurredspeech, scoliosis, diabetes, heart disorders (hypertrophiccardiomyopathy), elevated serum or plasma high sensitive troponin-T(hsTNT) (>14 ng/L), and reduced serum or plasma frataxin protein levels(≤19 ng/mL for pediatric and ≤21 ng/mL for adult patients). In someembodiments, the methods and compositions of the present technologyreduce the likelihood that a subject with risk factors for Friedreich'sataxia will develop one or more signs or symptoms of Friedreich'sataxia, or will delay the onset of Friedreich's ataxia.

In practicing the present technology, many conventional techniques inmolecular biology, protein biochemistry, cell biology, immunology,microbiology, and recombinant DNA are used. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ansubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, N Y, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively. Methods to detect and measurelevels of polypeptide gene expression products (i.e., gene translationlevel) are well-known in the art and include the use polypeptidedetection methods such as antibody detection and quantificationtechniques. (See also, Strachan & Read, Human Molecular Genetics, SecondEdition. (John Wiley and Sons, Inc., NY, 1999)).

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this present technologybelongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, the term “about” indicates values that may deviate up to1%, 5%, 10%, 15%, and in some cases up to 20% higher or lower than thevalue referred to, the deviation range including integer values, and, ifapplicable, non-integer values as well, constituting a continuous range.

As used herein, the “administration” of an agent to a subject includesany route of introducing or delivering to a subject a compound toperform its intended function. Administration can be carried out by anysuitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),intrathecally, topically, iontophoretically and the like. Administrationincludes self-administration and the administration by another.

The term “binds” refers to formation of a complex between two or moremolecules to a statistically greater degree than would be expected fornon-interacting molecules; complexes so formed may include covalentbonding or non-covalent bonding (e.g., hydrogen bonding) between two ormore of the molecules of the complex. Methods for the detection ofcomplexes involving DNA are well known in the art. Binding ischaracterized by a dissociation constant (K_(D)), well known in the art.

As used herein, “bromodomain” refers a portion of a polypeptide thatrecognizes acetylated lysine residues. In one embodiment, a bromodomainof a BET family member polypeptide comprises approximately 110 aminoacids and shares a conserved fold comprising a left-handed bundle offour alpha helices linked by diverse loop regions that interact withchromatin.

By “BET family polypeptide” is meant a polypeptide comprising twobromodomains and an extraterminal (ET) domain or a fragment thereofhaving transcriptional regulatory activity or acetylated lysine bindingactivity. Exemplary BET family members include Brd2, Brd3, Brd4 andBrdT. Brd4 is a member of the BET family of bromodomain-containingproteins that bind to acetylated histones to influence transcription. Asused herein, “Brd4 polypeptide” refers to a protein or fragment thereofhaving at least 85% identity to NP_055114 that is capable of bindingchromatin or regulating transcription. The sequence (SEQ ID NO: 1) ofthe exemplary Brd4 polypeptide NP_055114 is shown below:

  1 msaesgpgtr lmlpvmgdg  letsqmsttq aqaqpqpana astnppppet snpnkpkrqt 61 nqlqyllrv  lktlwkhqfa wpfqqpvdav klnlpdyyki iktpmdmgti kkrlennyyw121 naqeciqdfn tmftncyiyn kpgddivlma ealeklflqk inelpteete imivqakgrg181 rgrketgtak pgvstvpntt qastppqtqt pqpnpppvqa tphpfpavtp dlivqtpvmt241 vppqplqtp  ppvppqpqpp papapqpvqs hppiiaatpq pvktkkgvkr kadtftptti301 dpiheppslp pepkttklgq rressrpvkp pkkdvpdsqq hpapeksskv seqlkccsgi361 lkemfakkha ayawpfykpv dvealglhdy cdiikhpmdm stiksklear eyrdaqefga421 dvrimfsncy kynppdhevv amarklqdvf emrfakmpde peepvvayss pavppptkvv481 appsssdsss dsssdsdsst ddseeeraqr laelqeqlka vheqlaalsq pqqnkpkkke541 kdkkekkkek hkrkeeveen kkskakeppp kktkknnssn snvskkepap mkskppptye601 seeedkckpm syeekrqlsl dinklpgekl grvvhiiqsr epslknsnpd eieidfetlk661 pstlrelery vtsclrkkrk pqaekvdvia gsskmkgfss sesesssess ssdsedsetg

As used herein, the term “Brd4 binding moiety” refers to compounds orsubportion(s) of an agent that are capable of specifically binding aBrd4 polypeptide.

As used herein, a “control” is an alternative sample used in anexperiment for comparison purpose. A control can be “positive” or“negative.”

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g. an amount that reduces, ameliorates, or delays the onset of thephysiological symptoms of Friedreich's ataxia. In the context oftherapeutic or prophylactic applications, in some embodiments, theamount of a composition administered to the subject will depend on thetype and severity of the disease and on the characteristics of theindividual, such as general health, age, sex, body weight, and toleranceto drugs. In some embodiments, it will also depend on the degree,severity, and type of disease. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors. Thecompositions can also be administered in combination with one or moreadditional therapeutic compounds. In the methods described herein,agents having a formula A-L-B, wherein -L- is a linker; A- is a Brd4binding moiety; and -B is a polyamide that specifically binds to one ormore repeats of a GAA oligonucleotide sequence, or a pharmaceuticallyacceptable salts thereof, such as acetate or trifluoroacetate salts, maybe administered to a subject having one or more signs, symptoms, or riskfactors of Friedreich's ataxia, such as, e.g., ataxia, gait ataxia,muscle weakness, loss of coordination, loss of balance, lack of reflexesin lower limbs, loss of tendon reflexes, loss of ability to feelvibrations in lower limbs, loss of sensation in the extremities, loss ofupper body strength, weakness in the arms, spasticity, loss of tactilesensation, impairment of position sense, impaired perception of lighttouch, impaired perception of pain, impaired perception of temperature,vision impairment, color vision changes, involuntary eye movements, pescavus, inversion of the feet, hearing impairment, dysarthria, dysphagia,impaired breathing, scoliosis, diabetes, glucose intolerance,carbohydrate intolerance, hypertrophic cardiomyopathy, arrhythmia,myocardial fibrosis, cardiac failure, elevated serum or plasma highsensitive troponin-T (hsTNT) (>14 ng/L), and reduced serum or plasmafrataxin protein levels (≤19 ng/mL for pediatric and ≤21 ng/mL for adultpatients). For example, a “therapeutically effective amount” of agentshaving a formula A-L-B wherein -L- is a linker; A- is a Brd4 bindingmoiety; and -B is a polyamide that specifically binds to one or morerepeats of a GAA oligonucleotide sequence includes levels at which thepresence, frequency, or severity of one or more signs, symptoms, or riskfactors of Friedreich's ataxia are reduced or eliminated. In someembodiments, a therapeutically effective amount reduces or amelioratesthe physiological effects of Friedreich's ataxia, and/or the riskfactors of Friedreich's ataxia, and/or delays the progression or onsetof Friedreich's ataxia.

As used herein, the terms “expansion” and “repeat expansion” refer tothe presence of contiguously repeated oligonucleotide sequences in agene. The term “hyper-expansion” refers to a level of expansion greaterthan typically observed in a population. For example, whereas typicalalleles may have an expansion of 6-34 repeats, a hyper-expanded allelemay include from 66-1700 repeats, or even more.

As used herein, “expression” includes, but is not limited to one or moreof the following: transcription of the gene into precursor mRNA;splicing and other processing of the precursor mRNA to produce maturemRNA; mRNA stability; translation of the mature mRNA into protein(including codon usage and tRNA availability); and glycosylation and/orother modifications of the translation product, if required for properexpression and function.

As used herein, the term “oligonucleotide sequence” refers to aplurality of nucleic acids having a defined sequence and length (e.g.,2, 3, 4, 5, 6, or even more nucleotides). Oligonucleotide sequences maybe present, for example, within genomic nucleotide sequences.Oligonucleotide sequences may also be present, for example, withinnucleotide sequences of genes. Oligonucleotide sequences may also bepresent, for example, within recombinant nucleotide sequences such asplasmids or vectors. The term “oligonucleotide repeat sequence” or“repeats of an oligonucleotide sequence” refers to a contiguousexpansion of oligonucleotide sequences. The terms “nucleic acid” and“nucleotide” refer to ribonucleotide and deoxyribonucleotide, andanalogs thereof, well known in the art.

As used herein, the term “predisposed to having” Friedreich's ataxiarefers to subjects with a family history of Friedreich's ataxia suchthat there is a possibility that the subject has inherited one or moregenetic loci comprising disease loci and will at some point develop adiagnosable disorder. The term also encompasses subjects heterozygous orhomozygous at a single disease locus or multiple disease loci.

As used herein, “specifically binds” means a compound or agent thatrecognizes and binds a particular polypeptide, polynucleotide, oroligonucleotide, but which does not substantially recognize and bindother molecules in a sample, for example, a biological sample, whichnaturally includes the particular polypeptide, polynucleotide, oroligonucleotide.

The term “suspected of having” refers to subjects who present withclinical or biochemical symptoms associated with Freidreich's ataxia,regardless of whether they have been diagnosed as having the disorder.

As used herein, the term “subject” refers to an organism administeredone or more compositions of the present technology. Typically, thesubject is a mammal, such as an animal, e.g., domestic animals (e.g.,dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horsesand the like) and laboratory animals (e.g., monkey, rats, mice, rabbits,guinea pigs and the like). In some embodiments, the subject is a human.

The term “transcription,” well known in the art, refers to the synthesisof RNA (i.e., ribonucleic acid) by DNA-directed RNA polymerase. The term“modulate transcription” and similar terms refer to a change intranscriptional level that can be measured by methods well known in theart, e.g., methods directed at the assay of mRNA, the product oftranscription. In some embodiments, modulation is an increase intranscription. In other embodiments, modulation is a decrease intranscription.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to preventing or ameliorating a disorder or condition and/orsymptoms associated therewith. By “ameliorate” is meant decrease,suppress, attenuate, diminish, arrest, or stabilize the development orprogression of a disease or condition. It will be appreciated that,although not precluded, treating a disorder or condition does notrequire that the disorder, condition or symptoms associated therewith becompletely eliminated. As used herein, the terms “prevent,”“preventing,” “prevention,” “prophylactic treatment” and the like referto reducing the probability of developing a disorder or condition in asubject, who does not have, but is at risk of or susceptible todeveloping a disorder or condition.

As referred to herein, in the therapeutic treatment of Friedreich'sataxia, the object is typically to reduce, alleviate or slow down(lessen) the pathologic condition or disorder. By way of example, butnot by way of limitation, a subject is successfully “treated” forFriedreich's ataxia if, after receiving a therapeutic amount of an agenthaving a formula A-L-B, wherein -L- is a linker; A- is a Brd4 bindingmoiety; and -B is a polyamide that specifically binds to one or morerepeats of a GAA oligonucleotide sequence, or a pharmaceuticallyacceptable salt thereof, such as acetate or trifluoroacetate salt,according to the methods described herein, the subject shows observableand/or measurable reduction in or absence of one or more signs andsymptoms of Friedreich's ataxia, such as but not limited to, e.g.,ataxia, gait ataxia, muscle weakness, loss of coordination, loss ofbalance, lack of reflexes in lower limbs, loss of tendon reflexes, lossof ability to feel vibrations in lower limbs, loss of sensation in theextremities, loss of upper body strength, weakness in the arms,spasticity, loss of tactile sensation, impairment of position sense,impaired perception of light touch, impaired perception of pain,impaired perception of temperature, vision impairment, color visionchanges, involuntary eye movements, pes cavus, inversion of the feet,hearing impairment, dysarthria, dysphagia, impaired breathing,scoliosis, diabetes, glucose intolerance, carbohydrate intolerance,hypertrophic cardiomyopathy, arrhythmia, myocardial fibrosis, cardiacfailure, elevated serum or plasma high sensitive troponin-T (hsTNT) (>14ng/L), and reduced serum or plasma frataxin levels (≤19 ng/mL forpediatric and ≤21 ng/mL for adult patients). It is also to beappreciated that the various modes of treatment of medical conditions asdescribed are intended to mean “substantial,” which includes total butalso less than total treatment, and wherein some biologically ormedically relevant result is achieved. Treating Friedreich's ataxia, asused herein, also refers to treating the signs and symptoms related toreduced frataxin activity or frataxin expression levels characteristicof Friedreich's ataxia. For example, treating Friedreich's ataxia mayrefer to increasing frataxin mRNA levels in a patient havingFriedreich's ataxia relative to the patient prior to treatment. TreatingFriedreich's ataxia may also refer to increasing frataxin protein levelsin a patient having Friedreich's ataxia relative to the patient prior totreatment.

As used herein, “Friedreich's ataxia” (FA or FRDA) refers to a diseaseor condition such that a subject has reduced levels of frataxin relativeto an unaffected subject or a healthy carrier having a Friedreich'sataxia (FRDA)-associated allele and where the subject has at least about50 GAA trinucleotide repeats in the frataxin (FXN) gene.

As used herein, a “triplet repeat” or “trinucleotide repeat” refers to apolymeric form of deoxyribonucleic acid (DNA) comprising a sequence unitthat is three nucleotides in length such that each sequence unit ismultiply repeated in a contiguous region in a gene. For example, a“triplet repeat” or “trinucleotide repeat” includes GAA trinucleotiderepeats that may be found in the frataxin gene.

Generally, reference to a certain element such as hydrogen or H is meantto include all isotopes of that element. For example, if an R group isdefined to include hydrogen or H, it also includes deuterium andtritium. Compounds comprising radioisotopes such as tritium, C¹⁴, P³²and S³⁵ are thus within the scope of the present technology. Proceduresfor inserting such labels into the compounds of the present technologywill be readily apparent to those skilled in the art based on thedisclosure herein.

In general, “substituted” refers to an organic group as defined below(e.g., an alkyl, cycloalkyl, and aryl groups) in which one or more bondsto a hydrogen atom contained therein are replaced by a bond tonon-hydrogen atoms. Substituted groups also include groups in which oneor more bonds to a carbon(s) or hydrogen(s) atom are replaced by one ormore bonds, including double or triple bonds, to a heteroatom. Thus, asubstituted group may be substituted with one or more substituents,unless otherwise specified. Examples of substituent groups include:halogens (i.e., F, Cl, Br, and I); alkyl, alkenyl, alkynyl, cycloalkyl,aryl, heteroaryl; hydroxyls; alkoxy, alkenoxy, aryloxy, cycloalkyloxy,aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, andheterocyclylalkoxy groups; carbonyls (oxo); carboxylates; esters;urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols;sulfides; sulfoxides; sulfones; sulfonyls; pentafluorosulfanyl (i.e.,SFs), sulfonamides; amines; N-oxides; hydrazines; hydrazides;hydrazones; azides; amides; ureas; amidines; guanidines; enamines;imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines;nitro groups; nitriles (i.e., CN); and the like. Alkyl, cycloalkyl,cycloalkylalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclyl,heteroaralkyl, alkoxy, amido, and amino groups may all be substitutedone or more times with non-hydrogen groups. If possible, groups may besubstituted at the alkyl, cycloalkyl, aryl, heterocyclyl, and/orheteroaryl group(s) of the group.

Alkyl groups include straight chain and branched chain alkyl groupshaving from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or,in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms.Examples of straight chain alkyl groups include groups such as methyl,ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octylgroups. Examples of branched alkyl groups include, but are not limitedto, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl,and 2,2-dimethylpropyl groups. Exemplary substituted alkyl groupsinclude, but are not limited to, halogenated alkyl (e.g.,trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl,dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like.

Cycloalkyl groups include mono-, bi- or tricyclic alkyl groups havingfrom 3 to 12 carbon atoms in the ring(s), or, in some embodiments, 3 to10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Exemplary monocycliccycloalkyl groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.In some embodiments, the cycloalkyl group has 3 to 8 ring members,whereas in other embodiments the number of ring carbon atoms range from3 to 5, 3 to 6, or 3 to 7. Bi- and tricyclic ring systems include bothbridged cycloalkyl groups and fused rings.

Cycloalkylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to acycloalkyl group as defined above. In some embodiments, cycloalkylalkylgroups have from 4 to 16 carbon atoms, 4 to 12 carbon atoms, andtypically 4 to 10 carbon atoms.

Alkenyl groups include straight and branched chain alkyl groups asdefined above, except that at least one double bond exists between twocarbon atoms. Alkenyl groups may have from 2 to 12 carbon atoms, andtypically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group hasone, two, or three carbon-carbon double bonds.

Alkynyl groups include straight and branched chain alkyl groups asdefined above, except that at least one triple bond exists between twocarbon atoms. Alkynyl groups have from 2 to 12 carbon atoms, andtypically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2to 6, or 2 to 4 carbon atoms. In some embodiments, the alkynyl group hasone, two, or three carbon-carbon triple bonds.

Aryl groups are cyclic aromatic hydrocarbons that do not containheteroatoms. Aryl groups herein include monocyclic, bicyclic andtricyclic ring systems. Thus, aryl groups include, but are not limitedto, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl,anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In someembodiments, aryl groups contain 6-14 carbons, and in others from 6 to12 or even 6-10 carbon atoms in the ring portions of the groups. In someembodiments, the aryl groups are phenyl or naphthyl.

Aralkyl groups are alkyl groups as defined above in which a hydrogen orcarbon bond of an alkyl group is replaced with a bond to an aryl groupas defined above. In some embodiments, aralkyl groups contain 7 to 16carbon atoms, 7 to 14 carbon atoms, or 7 to 10 carbon atoms. Exemplaryaralkyl groups include, but are not limited to, benzyl and phenethylgroups.

Heterocyclyl groups include aromatic (also referred to as heteroaryl)and non-aromatic (also referred to as heterocycloalkyl orheterocyclylalkyl) ring compounds containing 3 or more ring members, ofwhich one or more is a heteroatom such as, but not limited to, N, O, andS. In some embodiments, the heterocyclyl group contains 1, 2, 3 or 4heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi-and tricyclic rings having 3 to 16 ring members. Heterocyclyl groupsencompass aromatic, partially unsaturated and saturated ring systems,such as, for example, imidazolyl, imidazolinyl and imidazolidinylgroups. The phrase “heterocyclyl group” includes fused ring speciesincluding those comprising fused aromatic and non-aromatic groups.However, the phrase does not include heterocyclyl groups that have othergroups, such as alkyl, oxo or halo groups, bonded to one of the ringmembers. Rather, these are referred to as “substituted heterocyclylgroups”. Heterocyclyl groups include, but are not limited to,aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl,furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl,pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl,thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl,piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl,tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl,pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl,dihydrodithiinyl, dihydrodithionyl, homopiperazinyl, quinuclidyl,indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl,indolizinyl, benzotriazolyl, benzimidazolyl, benzofuranyl,benzothiophenyl, benzthiazolyl, benzoxadiazolyl, benzoxazinyl,benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl,benzothiazolyl, benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl,imidazopyridyl (azabenzimidazolyl), triazolopyridyl, isoxazolopyridyl,purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl,quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl,naphthyridinyl, pteridinyl, thianaphthyl, dihydrobenzothiazinyl,dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl,tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl,tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl,tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl,tetrahydrotriazolopyridyl, tetrahydroquinolinyl, 1,2-diazepanyl,1,3-diazepanyl, and 1,4-diazepanyl groups. Exemplerary heterocyclylgroup include, but are not limited to, pyridyl and thiazolyl groups.

Heteroaralkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to aheteroaryl group as defined above.

Heteroatom as used herein refers to an atom of any element other thancarbon or hydrogen. Exemplary heteroatoms include, but are not limitedto, nitrogen, oxygen, sulfur, and phosphorus.

Groups described herein having two or more points of attachment (i.e.,divalent, trivalent, or polyvalent) within the compound of the presenttechnology are designated by use of the suffix, “ene.” For example,divalent alkyl groups are alkylene groups, divalent aryl groups arearylene groups, divalent heteroaryl groups are divalent heteroarylenegroups, and so forth.

Alkoxy groups are hydroxyl groups (—OH) in which the bond to thehydrogen atom is replaced by a bond to a carbon atom of an alkyl groupas defined above. Examples of linear alkoxy groups include but are notlimited to methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and thelike. Alkoxyalkyl groups are alkoxy groups in which the oxygen is bondedto both an alkyl group and an alkylene group.

The term “amide” (or “amido”) includes C- and N-amide groups, i.e.,—C(O)NR⁷¹R⁷², and —NR⁷¹C(O)R⁷² groups, respectively. R⁷¹ and R⁷² areindependently hydrogen, or a substituted or unsubstituted alkyl,alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl orheterocyclyl group as defined herein.

The term “nitrile” or “cyano” as used herein refers to the —CN group.

The term “amine” (or “amino”) as used herein refers to —NR⁷⁵R⁷⁶ groups,wherein R⁷⁵ and R⁷⁶ are independently hydrogen, or a substituted orunsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl,heterocyclylalkyl or heterocyclyl group as defined herein. In someembodiments, the amine may be alkylamino, dialkylamino, arylamino, oralkylarylamino. In some embodiments, the amino group may be positivelycharged (e.g., —NH₃+).

The term “halogen” or “halo” as used herein refers to bromine, chlorine,fluorine, or iodine. In some embodiments, the halogen is fluorine. Inother embodiments, the halogen is chlorine or bromine.

The term “halogenated alkyl” or “haloalkyl” as used herein refers toalkyl groups as defined above that are mono-, di-, or polysubstituted byhalogen. Exemplary halogenated alkyl groups include, but are not limitedto, fluoromethyl and trifluoromethyl.

EXAMPLES

The present technology is further illustrated by the following examples,which should not be construed as limiting in any way.

Unless otherwise noted, starting materials were purchased from standardchemical suppliers and used without further purification. Water waspurified with a NANOPURE® water purification system (18.2 MΩ). Allbuffers were filtered (0.2 μm) before use. Oligonucleotide oligomerswere purchased from Integrated DNA Technologies Inc. Cell culture mediaand reagents were purchased from Invitrogen.

Example 1 Synthesis of Subunit -B Polyamide

Polyamide 1 (see Polyamide 1 formula below) and Polyamide 3 (seePolyamide 3 formula below) were synthesized by manual solid-phasesynthesis using Boc-beta-alanine PAM resin following establishedprocedures (Boc=tert-butoxycarbonyl) in Baird, E. E. et al., J. Am.Chem. Soc. 118, 6141-6146 (1996), which is incorporated herein byreference. After synthesis was complete, the polyamides were cleavedfrom the support by aminolysis with 3,3′-diamino-N-methyldipropylamine(55° C., 12 h). The polyamides were precipitated twice with diethylether, dissolved in 15% acetonitrile/H₂O+0.1% trifluoroacetic acid(TFA), and purified by reverse-phase preparative HPLC on a C18 column.The clean polyamide fractions were frozen and lyophilized to afford awhite or off-white powder. The quantity of each polyamide was measuredby UV/Vis spectroscopy with a molar extinction coefficient of 8650 M⁻¹cm¹ at λ_(max) near 310 nm for each N-methylpyrrole, N-methylimidazole,or 3-chlorothiophene.

Table 1 illustrates the identity and purity of control polyamides 1 and3 using MALDI-TOF mass spectrometry and analytical HPLC.

TABLE 1 Mass spectrometric of polyamides 1 and 3. Mass MoleculeMolecular formula Mass Calculated Observed Control polyamide 1C₅₉H₇₃ClN₂₁O₁₀S 1302.53 1303.14 Control polyamide 3 C₄₃H₆₁N₁₈O₈ 957.49958.11

Example 2 Synthesis of A-L-B Agent

Exemplary subunit A-, (S)-JQ1, was synthesized as previously describedin Filippakopoulos, P. et al., Nature 468, 1067-1073 (2010), which isincorporated herein by reference. As shown in Scheme 1, following thesynthesis of (S)-JQ1, the tert-butyl group was hydrolyzed with formicacid (23° C., 72 h) to afford (S)-JQ1 acid. The JQ1 acid was thenactivated using 1-Hydroxy-7-azabenzotriazole (HOAt) and1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU), then coupled to linkerH₂N-PEG6-CH₂CH₂COOH (23° C., 4 h) to afford (S)-JQ1-PEG6. The resultingmolecule was purified by reverse-phase HPLC and verified by MALDI-TOFmass spectrometry. Fractions that showed clean molecule were frozen andlyophilized to afford an oil.

Table 2 illustrates the identity and purity of (S)-JQ1-PEG6 usingMALDI-TOF mass spectrometry and analytical HPLC.

TABLE 2 Mass spectrometric of (S)-JQ1-PEG6. Molecule Molecular formulaMass Calculated Mass Observed (S)-JQ1-PEG6 C₃₄H₄₇ClN₅O₉S⁺ 736.28 736.95

As shown in Scheme 2, (S)-JQ1-PEG6 was activated with HOAt and HATU inN,N-diisopropyethylamine (DIEA) and dimethylformamide (DMF), thencoupled to control polyamides 1 or 3 to yield the control polyamide1-PEG6-JQ1 (control conjugate 1) and control polyamide 3-PEG6-JQ1 (Agent4), respectively. The conjugates were purified by reverse-phase HPLC andanalyzed by MALDI-TOF mass spectrometry (Table 3). Fractions that showedclean conjugate without contaminants were frozen in liquid nitrogen andlyophilized to afford a white or off white powder.

Table 3 illustrates the identity and purity of control conjugate 2 andAgent 4 using MALDI-TOF mass spectrometry and analytical HPLC.

TABLE 3 Mass spectrometric of control conjugate 2 and Agent 4. Mass MassMolecule Molecular formula Calculated Observed Control conjugate 2C₉₃H₁₁₇Cl₂N₂₆O₁₈S₂ 2019.79 2020.27 Agent 4 C₇₇H₁₀₅ClN₂₃O₁₆S 1674.751675.32

Example 3 Agents of the Present Technology Increase Frataxin Levels inFRDA Patient Cells

A. Cell Culture

GM15850 and GM15851 cell lines (Epstein Barr virus transformed humanB-lymphocytes) were obtained from the National Institute of GeneralMedical Sciences (NIGMS) Human Genetic Cell Repository at the CoriellInstitute for Medical Research, Camden, N.J. The GM15850 cell line wasderived from a clinically affected 13 year old Friedreich's ataxiapatient ataxia displaying scoliosis, hypertrophic cardiomyopathy, andslurred speech, and characterized as homozygous for the GAA expansion inthe frataxin gene with alleles of approximately 650 and 1030 repeats.The GM15851 cell line was derived from a clinically unaffectedindividual having two FRDA alleles in the normal range of GAAtrinucleotide repeats (from about 6 to about 34 repeats). Thisclinically unaffected individual is a brother of the clinically affectedpatient from which the GM15850 cell line was derived.

Cells were maintained in RPMI 1640 supplemented with 2 mM L-glutamineand 15% fetal bovine serum (FBS) at 37° C. in a humidified atmosphere of5% CO₂. Cell density was maintained between 200,000 cells/mL and1,000,000 cells/mL. Cell growth and morphology were monitored by phasecontrast microscopy and cell viability by trypan blue exclusion. Tomaintain cell density, cells were passaged by centrifuging at 500g for 3minutes and resuspending the cells in fresh media. To avoidcross-contamination, cells were handled separately with separate bottlesof media. Relevant data were obtained from cell lines passaged betweenP9 and P12.

B. RT-PCR

To prepare agents for treatment of cells, each agent or agent mixturetested was dissolved in DMSO. The final concentration of DMSO was 0.1%except where JQ1 and Polyamide 3 combined were added to the same cells.For that treatment (JQ1+3), the final concentration of DMSO was 0.2%.500,000 cells ells were seeded at 500,000 cells/mL and treated 24 h withagents in DMSO (0.1-0.2% DMSO final concentration). For the 24 hourtreatment, cell media was changed immediately before treatment withagent and agent was added at the zero time point. The media was notchanged during the 24 hour treatment time period.

After treatment with the agents or agent mixtures, cells were harvestedand total RNA was purified with the RNeasy Mini Kit (Qiagen, Valencia,Calif.), including on-column DNase I treatment (ZYMO Research, Catalognumber E1010), according to manufacturer's directions. The optionaldrying step, according to the manufacturer's instructions, was performedprior to elution of the purified RNA, and the RNeasy spin column wasplaced in a new collection tube and centrifuged at full speed for 1 minto remove residual buffers. cDNA was synthesized from 250 ng purifiedRNA via the iScript cDNA synthesis kit according to manufacturer'sinstructions (Bio-Rad, Hercules, Calif.). qPCR was performed with iTaqUniversal SYBR Green Supermix (Bio-Rad) on a CFX Connect 96 instrument(Bio-Rad). cDNA was analyzed with PCR parameters as follows: 1 cycle of95° C. 2 min and 40 cycles of 95° C. 5 s, 54° C. 30 s. Primer pairs forTATA-box binding protein (TBP) and FXNwere used. The following primerswere used for TBP: 5′-CCACTCACAGACTCTCACAAC-3′ (forward) (SEQ ID NO: 16)and 5′-CTGCGGTACAATCCCAGAACT-3′ (reverse) (SEQ ID NO: 17). The followingprimers for FXN were: 5′-AGCCAGATTTGCTTGTTTGG-3′ (forward) (SEQ ID NO:18) and 5′-CAGAGGAAACGCTGGACTCT-3′ (reverse) (SEQ ID NO: 19). Thefollowing primers were used for BRWD1: 5′-CCAGCGCATCGGTCCTAT-3′(forward) (SEQ ID NO: 20) and 5′-CTTCCTGCACCAAGTAAAGAAGT-3′ (reverse)(SEQ ID NO: 21). Expression was normalized to TBP. Error bars representstandard error of the mean for n=4 biological replicates.

C. Results

Agent 4 increases frataxin (FXN) mRNA levels in a cell line derived froma patient with Friedreich's ataxia (FRDA). As shown in FIG. 1A, frataxin(FXN) mRNA was measured by RT-PCR, relative to that of TATAA-box bindingprotein (TBP) mRNA, in a cell line derived from a patient withFriedreich's ataxia (GM15850). 24 hour treatment of FRDA patient cells(GM15850) with 0.1% DMSO served as a control. Additional controltreatments for 24 hours with 0.1% DMSO, 1 μM Polyamide 3, 1 μMunconjugated JQ1, 1 μM Polyamide 3 and 1 μM unconjugated JQ1(combination treatment), or 1 μM Control Conjugate 2 resulted in noincrease in FXN mRNA levels. Treatment for 24 hours with 100 nM Agent 4resulted in a slight (1.77-fold) increase in FXN mRNA levels. Treatmentfor 24 hours with 500 nM Agent 4 resulted in a 4.2-fold increase in FXNmRNA levels. Surprisingly, treatment for 24 hours with 1 μM Agent 4resulted in an 8.5-fold increase in FXN mRNA levels. A 3-fold increasein FXN mRNA levels was observed after 6 hours treatment with 1 μM Agent4 (data not shown). As shown in FIG. 1B, no changes in FXN mRNA levelswere observed when treatment was performed using the GM15851 clinicallyunaffected cell line. As expected, the GM15850 FRDA patient cell linehad a lower level of FXN mRNA compared to the GM15851 clinicallyunaffected cell line.

Example 4 Luciferase Reporter Assay

The cellular consequence of Agent 4 on frataxin (FXN) expression wasexamined using a luciferase reporter assay. The luciferase reporterassay is described in Lufino et al., Hum. Mol. Genet., 22(25): 5173-5187(2013), the disclosure of which is herein incorporated by reference.This reporter, which includes a ˜310 GAA repeat, recapitulates both thetranscriptional repression and the heterochromatin formation at theendogenous FXN locus that are hallmarks of the FRDA disease. After 24hours of treatment with Agent 4 (at 1 or 2 μM), or control treatments(Control Conjugate 2 (“2”), Polyamide 3 (“3”) or JQ1), cells wereharvested and luciferase activity was measured. FIG. 2 shows a bar graphof luciferase activity for treated reporter cell lines FAN-Luc andFXN-GAA-Luc. The FXN-GAA-Luc line, containing ˜310 GAA repeats in thefirst intron of FXN, was treated for 24 hours with the indicatedmolecules. Treatments are 2 μM unless otherwise indicated. Results aremean±SEM (n=4). The FXN-Luc line, containing 6 GAA repeats, was treatedwith the same conditions as for the FXN-GAA-Luc line.

A dose-dependent increase in luciferase activity was observed aftertreatment with Agent 4, exceeding the activity of Compound 109, ahistone deacetylase inhibitor that recently completed a Phase Ibclinical trial for the treatment of Friedreich's ataxia (see Jacoby, D.et al., (PL1.003) Neurology, 82 (2014). The chemical structure ofCompound 109 is shown below.

Significant activation was not observed for any of the controltreatments, including treatment with Polyamide 3, JQ1, and theunconjugated combination of Polyamide 3 and JQ1. As an orthogonal testof specificity, we profiled the same compounds in a reporter cell linewith just 6 GAA repeats in the first intron of FXN. Neither Agent 4, norany of the controls, increased luciferase activity in the control line,confirming that the mechanism of action for Agent 4 is dependent on theGAA-repeat expansion (See FIG. 2). These results confirm that apolyamide-JQ1 conjugate can induce a robust increase of frataxinexpression.

Methods

Cells containing FXN-Luc or FXN-GAA-Luc were seeded onto 24-well platesat a confluency of 20%. The next day, media was refreshed and cells weretreated 24 hours with the indicated compound at the indicatedconcentration. Next, cell lysates were harvested and analyzed with theLuciferase Assay System (Promega, Madison, Wis.) per manufacturer'sdirections on a Synergy H4 Hybrid Reader (BioTek, Winooski, Vt.).

Example 5 Immunoblot Assay

To assess the effects of treatment with a polyamide-JQ1 conjugate (e.g.,Agent 4) on FXN protein, endogenous FXN levels were measured byimmunoblot. After 24 hours of treatment, a dose-dependent increase inFXN protein was observed in GM15850 cells treated with Agent 4 (FIG.3A). Neither polyamide alone (3), nor JQ1, nor the unconjugatedtreatment induced increased production of FXN protein. Furthermore, wedid not detect overt increase in FXN protein in the unaffected GM15851cells treated with Agent 4, consistent with this molecule targeting GAArepeats (FIG. 3B).

FIGS. 3A and 3B show immunoblots for FXN and α-tubulin (TUB) in GM15850cells (FIG. 3A) or GM15851 cells (FIG. 3B) treated with varyingconcentrations of Control Conjugate 2 (“2”), Polyamide 3 (“3”), JQ1 orAgent 4 (“4”). In FIG. 3A, “50” refers to GM15850 cells and in FIG. 3B,“51” refers to GM15851 cells. Cells were treated as in Example 4. Alltreatments are 24 hours in duration with 1 μM of the indicated molecule,except DMSO (0.1%) and Agent 4 (0.1, 0.5, or 1 μM). The results confirman increase in FXN protein after treatment of GM15850 cells with Agent 4and treatment of GM15851 cells with Agent 4 or Control Conjugate 2.

Methods

Total cell extracts from cell lines were used for immunoblot analysis.Antibodies to frataxin (abcam ab110328, 1:250 dilution) or alpha-Tubulin(Cell Signaling Technologies 2144, 1:1000 dilution) were used. Signalwas detected by chemiluminescence with HRP-conjugated secondaryantibodies (Bio-Rad 172-1011, 1:2000 for anti-mouse frataxin and GENA934V, 1:2000 for anti-rabbit alpha-Tubulin).

Example 6 mRNA Levels Measure Viability

To assess the effects of treatment with the compositions of the presenttechnology on cell viability, RNA concentrations of GM15850 cells weremeasured after 24 hours of treatment with each molecule (Controlconjugate 2 (“2”), unconjugated JQ1-(S), Control polyamide 3 (“3”),Control polyamide 3 and JQ1-(S) (unconjugated), Agent 4 (“4”), andControl polyamide 3 bound to JQ1 without a linker (“3-JQ1-(S)”)).

FIG. 4 is a graph showing relative RNA concentrations of cells harvestedafter 24 hours of treatment with each of the molecules described above.The results show that, without a linker, 3-JQ1-(S) is cytotoxic. Theresults also show that cells treated with the conjugate molecules (Agent4) have higher RNA concentrations than those treated with JQ1-(S) orunconjugated Control polyamide 3+JQ1-(S). JQ1-(S) is known to cause cellcycle arrest.

Methods

GM15850 cells were passaged into fresh medium at a concentration of500,000 cells/mL. The cells were seeded into a 24-well plate and treatedwith the described compounds for 24 hours in quadruplicate. After 24hours, the cells were collected, media was removed, and RNA washarvested with a Qiagen RNeasy Mini-Kit (Qiagen, Valencia, Calif.). RNAconcentrations were then measured by nanodrop UV-Vis spectrophotometry.

Example 7 Lymphoblastoid Cell Line (LCL) Data

To assess the effects of treatment with the compositions of the presenttechnology on frataxin (FXN) mRNA levels in lymphoblastoid cells, mRNAlevels from three different lymphoblastoid cell lines (LCL) derived fromFRDA patient samples were measured after 24 hours of treatment with theControl polyamide 3 and JQ1 (unconjugated) (“3+JQI”) or Agent 4 (“4”).

FIG. 6 is a graph showing relative frataxin (FXN) mRNA levels from threedifferent lymphoblastoid cell lines that were derived from patientsamples (LCL-P1, LCL-P2, and LCL-P3). Treatments were fore 24 hours withthe described molecules. The results demonstrate an increase in FXN mRNAafter treatment of lymphoblastoid cells from FRDA patients with Agent 4.

Methods

FRDA patients (n=3), LCL generation was carried out by Epstein BarrVirus (EBV) induced transduction of B cells, isolated from healthyindividuals and patients. The following steps were taken: (a) 2−3×10⁶cells/ml were seeded into T25 flask (Nunc) in RPMI 1640 medium(Hlimedia) containing 15% FBS (Gibco), 2 mM glutamine (Invitrogen) withthe presence of antibiotics, Penicillin (100 I.U./mL) and Streptomycin(100 μg/mL) (Invitrogen); (b) 3 mL of EBV supernatant was added in theflask and kept upright for 3 hours in an incubator (Eppendort) set at37° C. with 5% CO₂; (c) after 3 hours, 3 mL of 20% RPMI-1640 containing2 ug/mL of cyclosporine A (Sigma) was added and incubated upright for 6days without feeding (cells started showing small clumps and changes inmorphology from the 5-6th day onwards depending on the cell line); (d)on the 6th day complete medium change was done by spinning down cells at1000 rpm for 3 minutes and re-suspending the pellet in 15% freshcomplete RPML medium; and (e) after 2-3 weeks of incubation, appearanceof rosette/clump morphology of cells indicate the transformed phenotypeof PBMCs.

The treatments and data collection were performed as follows. Compoundtreatments were given in quadruplicate. Each cell line was furtherdivided into DMSO/Unconjugated/Conjugate categories (Total 6×10⁶ cellswere seeded in 12-well plate (Nunc, Delta Surface) at density of 0.5×10⁶cells per well in a final volume of 1 mL medium). In vehicle control(DMSO treated group) 1.0 μL DMSO (Sigma) i.e., 0.1% v/v was added,whereas for Unconjugated and Conjugate categories the finalconcentration of respective compounds were kept 1.0 μM. After 24 hoursof incubation, treated cells were further taken for RNA isolation. RNAwas harvested with Qiagen RNeasy mini-kit per manufacturer'sinstructions. RT was done with Applied Biosystem's cDNA synthesis kit.Standard qPCR was performed, normalizing to GAPDH.

Example 8 Peripheral Blood Mononuclear (PBMC) Data

To assess the effects of treatment with the compositions of the presenttechnology on frataxin (FXN) mRNA levels in primary patient peripheralblood mononuclear cells (PBMCs), mRNA levels from eleven different PBMCsamples derived from FRDA patients (P1-P11) were measured after 24 hoursof treatment with Agent 4 (“4”) (FIG. 7).

FIG. 7 is a graph showing relative FXN mRNA levels from eleven differentPBMC samples derived from FRDA patients following 24-hour treatment withAgent 4. The results demonstrate an increase in FXN mRNA after treatmentof PBMCs from FRDA patients with Agent 4.

Methods

PBMC isolation was done using Ficoll Histopaque gradient (Sigma-Aldrich)and the number of cells present were counted using hemocytometer.Peripheral blood was drawn from FRDA patients into ACD Buffer vial andstored at room temperature. Biological safety cabinet (ESCO Class IIBSC) and required materials were cleaned thoroughly with 70% ethanol(Merck KGaA). Meanwhile, 1×DPBS (Gibco Life technology Ref. 14190-144),Ficoll Histopaque (Sigma Lot#RNBF-2365), 15% RPMI-1640 (HIMEDIARef.-AL060), HIFBS (Gibco Cat. no. 10082147) were warmed in water bathset at 37° C. (Sun Scientific Industries). 8 mL of blood was dilutedwith 8 mL 1×DPBS (1:1), and mixed properly to make it a homogeneoussolution. Carefully, 8 mL diluted blood cell suspension was layered over4 mL of Histopaque (1:2 ratio) in a 15 mL falcon tube. The falcons werespun at 400×g for 40 minutes in swing out bucket rotor without break(Heraceus Megafuge-16R Centrifuge), at room temperature. Aftercentrifugation, the upper layer leaving the whitish buffy coat(lymphocyte layer) was transferred into new 15 mL falcon and brought upto 4 mL with 1×DPBS. The falcons were spun at 400×g for 12 minutes withbreaks on. The previous step was repeated. The supernatant was discardedand cell pellet was re-suspended in 15% RPMI. Cell count was performedusing Hemocytometer.

The treatments and data collection were performed as follows. Compoundtreatments were given in quadruplicate. Each cell line was furtherdivided into DMSO/Unconjugated/Conjugate categories (Total 6×10⁶ cellswere seeded in 12-well plate (Nunc, Delta Surface) at density of 0.5×10⁶cells per well in a final volume of 1 mL medium). In vehicle control(DMSO treated group) 1.0 μL DMSO (Sigma) i.e., 0.1% v/v was added,whereas for Unconjugated and Conjugate categories the finalconcentration of respective compounds were kept 1.0 μM. After 24 hoursof incubation, treated cells were further taken for RNA isolation. RNAwas harvested with Qiagen RNeasy mini-kit per manufacturer'sinstructions. RT was done with Applied Biosystem's cDNA synthesis kit.Standard qPCR was performed, normalizing to GAPDH.

Example 9 Binding of the Agents of the Present Technology to Brd4

To confirm that the conjugates of the present technology bind to Brd4,an AlphaScreen™ assay (Amplified Luminescent Proximity Homogeneousassay) was performed. FIG. 8 is a graph showing the binding of themolecules of the present technology to Brd4 (the target of JQ1-(S)). Theresults demonstrate that the conjugates bind to Brd4 as well as JQ1-(S)does independently.

Methods

The AlphaScreen™ assay is a bead-based proximity assay. There areacceptor and donor beads that, when proximal, amplify a chemiluminescentsignal via reaction with a singlet oxygen species. For this assay, Brd4and acetylated lysine are each bound to one type of bead. Then, ascompounds are titrated, signal disappears due to out competing theacetylated lysine residues. Compounds ranged in concentration from 10 nMto 1 μM.

Example 10 ChIP-Sequencing at the FXN Locus

To confirm that the conjugates of the present technology recruit Brd4,the super elongation complex, and a Cdk9 kinase that phosphorylates Ser2of the CTD of RNA Polymerase II, a series of ChIP-seq experiments wereperformed. FIG. 9C shows an FXN locus increase in Brd4 and FIG. 10Cshows an FXN locus increase in pSer2 occupancy in the Agent 4-treatedGM15850 cells relative to DMSO-treated GM15850 cells (FIGS. 9A and 10A)and 3+JQ1-(S)-treated GM15850 cells (FIGS. 9B and 10B). In the case ofRNAPol2 (pol2), FIG. 11C, shows an increase in elongating RNAPol2 movingthrough the FXN locus gene body in the Agent 4-treated GM15850 cellsrelative to DMSO- and 3+JQ1-(S)-treated GM15850 cells (FIGS. 11A and11B, respectively).

Methods

This is an example of a typical ChIP-Rx experiment. It is ChIP-seq butwith a drosophila spike-in for better normalization. According tomethods known in the art, three different IPs were done (Total RNAPol2,phospho-Ser2 of the CTD of RNAPol2, and Brd4). Brd4 required more cells(˜10⁸ per treatment) compared to the others (˜2.5×10⁷). Three treatmentswere performed for 24 hours in GM15850 cells (DMSO, unconjugated3+JQ1-(S), and Agent 4, both at 1 μM). The plots shown in FIGS. 9A-9C,FIGS. 10A-10C, and FIGS. 11A-11C are of read density over the entirefrataxin gene body.

EQUIVALENTS

The present technology is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods andapparatuses within the scope of the present technology, in addition tothose enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presenttechnology is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this present technology is notlimited to particular methods, reagents, compounds compositions orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. As will also be understood by oneskilled in the art all language such as “up to,” “at least,” and thelike, include the number recited.

What is claimed is:
 1. An agent having a formula A-L-B wherein -L- is alinker; A- is a bromodomain-containing protein 4 (Brd4) binding moietyhaving a structure of a bromodomain inhibitor; and -B is a nucleic acidbinding moiety that specifically binds to one or more repeats of a GAAoligonucleotide sequence.
 2. The agent of claim 1, wherein the -B is apolyamide that specifically binds to one or more repeats of a GAAoligonucleotide sequence.
 3. The agent of claim 1, wherein the agent iscapable of increasing frataxin (FXN) mRNA levels in a GM15850Friedreich's ataxia (FRDA) patient cell line relative to an untreatedGM15850 cell.
 4. The agent of claim 1, wherein the -B comprises one ormore of the following subunits:

wherein Z is hydrogen, amino, or amido group.
 5. The agent of claim 4,wherein the -B comprises -X-(β-Py-Im)_(n)-β-Py-TRM; X is -β-Im-, -β-Py-,-β-, or a bond; n is 1-10; and -TRM is -ImT or -CTh.
 6. The agent ofclaim 4, wherein the -B comprises -X-(β-Py-Im)_(n)-β-Py-TRM; X is-β-Im-, -β-Py-, -β-, or a bond; n is 1-10; and -TRM is -ImT or -CTh;wherein one of the -β-Py-Im- trimers is replaced by a -β-Im-Im- trimer.7. The agent of claim 5, wherein the -B comprises-(β-Py-Im)_(n)-β-Py-ImT; Z is hydrogen; and n is 1 or
 2. 8. The agent ofclaim 5, wherein the -B comprises -β-Py-β-Py-Im-β-Py-ImT or-β-Py-β-Py-Im-β-Py-CTh; and Z is hydrogen.
 9. The agent of claim 1,wherein -L- is a linker having a backbone chain which comprises at leastabout 10 continuous atoms.
 10. The agent of claim 9, wherein -L-comprises a combination of one or more linking moieties selected fromthe group consisting of arylene, cycloalkylene, heteroarylene,heterocycloalkylene, —O—, —(CH₂)_(x)—, —(OCH₂CH₂)_(y)—, —C(O)NR′—,—NR′C(O)—, —C(O)—, —NR*—, —(CH₂CH₂CH₂O)_(y)—, —(OCH₂CH₂CH₂)_(y)—, and

wherein R′ and R* are each independently a hydrogen or C₁-C₆ alkyl; andx and y are each independently an integer from 1-10.
 11. The agent ofclaim 9, wherein -L- comprises—(CH₂)_(X)—C(O)NH—(CH₂CH₂O)_(Y)—(CH₂)_(Q)—C(O)NH—(CH₂)_(Z)—N(CH₃)—(CH₂)_(P)—NH—,—(CH₂)_(X)—C(O)NH—(CH₂)_(R)—(OCH₂CH₂)_(Y)—O—(CH₂)_(Q)—C(O)NH—(CH₂)_(Z)—N(CH₃)—(CH₂)_(P)—NH—,or—(CH₂)_(X)—C(O)NH—(CH₂)_(Z)—N(CH₃)—(CH₂)_(P)—C(O)NH—(CH₂CH₂O)_(Y)—(CH₂)_(Q)—NH—;x is an integer from 1 to 5; z, p, R and Q are each independently aninteger from 2 to 5; and y is an integer from 1 to
 10. 12. The agent ofclaim 9, wherein -L- comprises—(CH₂)—C(O)NH—(CH₂CH₂O)_(Y)—(CH₂)₂—C(O)NH—(CH₂)₃—N(CH₃)—(CH₂)₃—NH—; andy is an integer from 3 to
 10. 13. The agent of claim 9, wherein -L-comprises one or more linking moieties selected from (Gly-Ser-Gly)v and(Gly-Gly-Ser)w , where v and w are an integer from 1 to
 10. 14. Theagent of claim 1, wherein the A- is a triazolodiazepine Brd4 bindingmoiety.
 15. The agent of claim 14, wherein the A- is a triazolodiazepineBrd4 binding moiety having a formula

J is N, O or CR¹¹; K is N, O or CR¹¹; with the proviso that J and Kcannot both be —O—; P is N, except when one of J or K is O, P is C;wherein R¹¹ is a hydrogen or substituted or unsubstituted alkyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group; R¹ is ahydrogen or substituted or unsubstituted alkyl, hydroxyalkyl,aminoalkyl, alkoxyalkyl, halogenated alkyl, hydroxyl, alkoxy, or —COOR⁴;wherein R⁴ is a hydrogen, substituted or unsubstituted arylene,aralkylene, cycloalkylene, heteroarylene, heteroaralkylene,heterocycloalkylene, alkylene, alkenylene, alkynylene,cycloalkylalkylene, or cycloalkylalkylene group interrupted by one ormore heteroatoms; R² is a substituted or unsubstituted aryl, alkyl,cycloalkyl, or aralkyl group; R³ is a hydrogen, halogen, or substitutedor unsubstituted alkyl group; and Ring E is a substituted orunsubstituted aryl or heteroaryl ring.
 16. The agent of claim 14,wherein the A- is a thienotriazolodiazepine Brd4 binding moiety having aformula

wherein R³ is hydrogen or —CH₃; R¹, R⁵, and R⁷ are each independentlyhydrogen, methyl, ethyl, or halomethyl group; and R⁸ is a halogen. 17.The agent of claim 1, wherein -L- is a linker having a backbone chainwhich comprises about 15 to 30 continuous atoms; the -B comprises-X-(β-Py-Im)_(n)-β-Py-TRM; X is -β-Im-, -β-Py-, -β-, or a bond; n is 1or 2; -TRM is -ImT or -CTh; and the A- is a thienotriazolodiazepine Brd4binding moiety having a formula:

wherein R³ is hydrogen or -CH3; R¹, R⁵, and R⁷ are methyl; and R⁸ is ahalogen.
 18. The agent of claim 17, wherein the -L- is a linkercomprising—(CH₂)_(X)—C(O)NH—(CH₂CH₂O)_(Y)—(CH₂)_(Q)—C(O)NH—(CH₂)_(Z)—N(CH₃)—(CH₂)_(P)—NH—,—(CH₂)_(X)—C(O)NH—(CH₂)_(R)—(OCH₂CH₂)_(Y)—O—(CH₂)_(Q)—C(O)NH—(CH₂)_(Z)—N(CH₃)—(CH₂)_(P)—NH—or—(CH₂)_(X)—C(O)NH—(CH₂)_(Z)—N(CH₃)—(CH₂)_(P)—C(O)NH—(CH₂CH₂O)_(Y)—(CH₂)_(Q)—NH—;x is an integer from 1 to 5; z, p, R and Q are each independently aninteger from 2 to 5; and y is an integer from 1 to
 10. 19. The agent ofclaim 17, wherein the -B comprises a polyamide sequence selected fromthe group consisting of: β-Im-β-Py-Im-β-Py-ImT, β-Py-β-Py-Im-β-Py-ImT,β-β-Py-Im-β-Py-ImT, β-Py-Im-β-Py-ImT, β-Im-β-Py-Im-β-Py-Im-β-Py-ImT,β-Py-β-Py-Im-β-Py-Im-β-Py-ImT, β-Py-Im-β-Py-Im-β-Py-ImT,-β-β-Py-Im-β-Py-Im-β-Py-ImT, β-Py-Im-β-Py-CTh and β-Py-β-Py-Im-β-Py-CTh.


20. A pharmaceutical composition comprising a therapeutically effectiveamount of the agent of claim 1 and a pharmaceutically acceptablecarrier.
 21. A method for increasing frataxin (FXN) mRNA levels in acell comprising contacting the cell with an effective amount of theagent of claim
 1. 22. The method of claim 21, wherein the cell comprisesa frataxin (FXN) gene including at least about 30 GAA repeats.
 23. Themethod of claim 21, wherein the cell comprises a gene, which includes atleast about 30 GAA repeats and a sequence encoding a functional frataxinpolypeptide sequence fused to a reporter gene.
 24. A method forincreasing frataxin (FXN) protein levels in a cell, comprisingcontacting the cell with an effective amount of the agent of claim 1.25. The method of claim 24, wherein the cell comprises frataxin (FXN)gene including at least about 30 GAA repeats.
 26. The method of claim25, wherein the cell comprises a reporter gene fused to the 3′-end ofthe frataxin (FXN) gene.
 27. A method of treating Friedreich's ataxia(FRDA) in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of the agent of claim
 1. 28.The method of claim 27, wherein frataxin (FXN) mRNA levels are increasedrelative to those in the subject prior to treatment.
 29. The method ofclaim 27, wherein frataxin (FXN) protein levels are increased relativeto those in the subject prior to treatment.
 30. The method of claim 27,wherein the treatment comprises ameliorating one or more symptoms ofFriedreich's ataxia (FRDA).
 31. A method for increasing levels offunctional frataxin polypeptide in a cell, comprising contacting thecell with an effective amount of the agent of claim 1; wherein the cellcomprises a fusion gene including at least about 30 GAA repeats and asequence encoding a functional frataxin polypeptide sequence fused to aheterologous polypeptide sequence.